Robust controlled-release formulations

ABSTRACT

The present invention relates to compositions forming a low viscosity mixture of: a. at least one diacyl glycerol and/or at least one tocopherol; b. at least one phospholipid component comprising phospholipids having i. polar head groups comprising more than 50% phosphatidyl ethanolamine, and ii. two acyl chains each independently having 16 to 20 carbons wherein at least one acyl chain has at least one unsaturation in the carbon chain, and there are no more than four unsaturations over two carbon chains; c. at least one biocompatible, oxygen containing, low viscosity organic solvent; wherein optionally at least one bioactive agent is dissolved or dispersed in the low viscosity mixture; and wherein the pre-formulation forms, or is capable of forming, at least one non-lamellar liquid crystalline phase structure upon contact with an aqueous fluid. The invention further relates to methods of treatment comprising administration of such compositions, and to pre-filled administration devices and kits containing the formulations.

FIELD

The present invention relates to formulation precursors(pre-formulations) comprising lipids that upon exposure to water oraqueous media, such as body fluids, spontaneously undergo at least onephase transition, thereby forming a controlled release matrix whichoptionally is bioadhesive.

BACKGROUND

Many bioactive agents including pharmaceuticals, nutrients, vitamins andso forth have a “functional window”. That is to say that there is arange of concentrations over which these agents can be observed toprovide some biological effect. Where the concentration in theappropriate part of the body (e.g. locally or as demonstrated by serumconcentration) falls below a certain level, no beneficial effect can beattributed to the agent. Similarly, there is generally an upperconcentration level above which no further benefit is derived byincreasing the concentration. In some cases increasing the concentrationabove a particular level results in undesirable or even dangerouseffects.

Some bioactive agents have a long biological half-life and/or a widefunctional window and thus may be administered occasionally, maintaininga functional biological concentration over a substantial period of time(e.g. 6 hours to several days). In other cases the rate of clearance ishigh and/or the functional window is narrow and thus to maintain abiological concentration within this window regular (or even continuous)doses of a small amount are required. This can be particularly difficultwhere non-oral routes of administration (e.g. parenteral administration)are desirable. Furthermore, in some circumstances, such as in thefitting of implants (e.g. joint replacements or oral implants) the areaof desired action may not remain accessible for repeated administration.In such cases a single administration must provide active agent at atherapeutic level over the whole period during which activity is needed.

Sustained activity is furthermore important in situations where aphysical soothing or barrier property is provided by a formulation. Insuch circumstances the biological effect may be provided by, forexample, the separation of a biological tissue from some undesirableagent or environment or by the provision of a soothing interface betweenthe tissue and its surroundings. Where compositions provide such abarrier or interfacial property, whether including a “drug” type activeagent or not, it is an advantage if the composition is sufficientlypermanent to allow a reasonable period between administrations.

Different methods have been used and proposed for the sustained releaseof biologically active agents. Such methods include slow-release, orallyadministered compositions, such as coated tablets, formulations designedfor gradual absorption, such as transdermal patches, and slow-releaseimplants such as “sticks” implanted under the skin.

One method by which the gradual release of a bioactive agent has beenproposed is a so-called “depot” injection. In this method, a bioactiveagent is formulated with carriers providing a gradual release of activeagent over a period of a number of hours, days, weeks, or even months.These are often based upon a degrading matrix which gradually degradesand/or disperses in the body to release the active agent.

The most common of the established methods of depot injection reliesupon a polymeric depot system. This is typically a biodegradable polymersuch as poly (lactic acid) (PLA) and/or poly (lactic-co-glycolic acid)(PLGA) and may be in the form of a solution in an organic solvent, apre-polymer mixed with an initiator, encapsulated polymer particles orpolymer microspheres. The polymer or polymer particles entrap the activeagent and are gradually degraded releasing the agent by slow diffusionand/or as the matrix is absorbed. Examples of such systems include thosedescribed in U.S. Pat. No. 4,938,763, U.S. Pat. No. 5,480,656 and U.S.Pat. No. 6,113,943 and can result in delivery of active agents over aperiod of up to several months. These systems do, however, have a numberof limitations including the complexity of manufacturing and difficultyin sterilising (especially the microspheres). The local irritationcaused by the lactic and/or glycolic acid which is released at theinjection site is also a noticeable drawback. There is also often quitea complex procedure to prepare the injection dose from the powderprecursor requiring reconstitution of the system before administrationto a subject e.g. by injection.

From a drug delivery point of view, polymer depot compositions also havethe disadvantage of accepting only relatively low drug loads and havinga “burst/lag” release profile. The nature of the polymeric matrix,especially when applied as a solution or pre-polymer, causes an initialburst of drug release when the composition is first administered. Thisis followed by a period of low release, while the degradation of thematrix begins, followed finally by an increase in the release rate tothe desired sustained profile. This burst/lag release profile can causethe in vivo concentration of active agent to burst above the functionalwindow immediately following administration, then drop back through thebottom of the functional window during the lag period before reaching asustained functional concentration. Evidently, from a functional andtoxicological point of view this burst/lag release profile isundesirable and could be dangerous. It may also limit the equilibriumconcentration which can be provided due to the danger of adverse effectsat the “peak” point.

Previous depot systems have been sought to address the problem of burstrelease. In particular, the use of hydrolysed polylactic acid and theinclusion of poly lactic acid-polyethylene glycol block copolymers havebeen proposed to provide the “low burst” polymeric system described inU.S. Pat. No. 6,113,943 and U.S. Pat. No. 6,630,115. These systemsprovide improved profiles but the burst/lag effect remains and they donot address other issues such as the irritation caused by the use ofpolymers producing acidic degradation products.

One alternative to the more established, polymer based, depot systems isto use a lipid-based slow release matrix comprising a liquid crystallinephase. Systems of this type have been proposed, for example, in U.S.Pat. No. 5,151,272, and WO2005/117830. Such compositions have manyadvantages and are potentially highly effective, but in some situationsit can be an advantage to have lipid based compositions that are evenlonger lasting, more resistant to chemical and/or enzymatic degradationand/or more physically robust than those proposed in the knownliterature.

The formation of non-lamellar phases in certain regions of theamphiphile (e.g. lipid)/water, amphiphile/oil and amphiphile/oil/waterphase diagrams is a well known phenomenon. Such phases includenon-lamellar liquid crystalline phases such as the cubic P, cubic D,cubic G, cubic micellar and hexagonal phases, which are fluid at themolecular level but show significant long-range order, and the L3 phasewhich comprises a multiply interconnected bi-continuous network ofbilayer sheets which are non-lamellar but lack the long-range order ofthe liquid crystalline phases. Depending upon the mean curvature of theamphiphile sheets or layers, these phases may be described as normal(mean curvature towards the apolar region) or reversed (mean curvaturetowards the polar region).

Knowledge of the spontaneous or preferred curvature of a particularcomponent allows some degree of prediction as to which structures willbe formed or formable by that amphiphile in aqueous mixtures. However,particularly where mixtures of amphiphiles is concerned, the exactnature of the phase structure and physical properties of the compositionwill depend greatly upon the specific interaction between the componentswith each other and/or with the solvent and other components of themixtures.

The non-lamellar liquid crystalline and L3 phases formed by certainamphiphiles and mixtures thereof are thermodynamically stable systems.That is to say, they are not simply a meta-stable state that willseparate and/or reform into layers, lamellar phases or the like, but arethe stable thermodynamic form of the lipid/solvent mixture.

The early attempts to develop lipid depot formulations, as in, forexample, U.S. Pat. No. 5,151,272 and U.S. Pat. No. 5,807,573, usingliquid crystal phases could in some cases be effective in terms ofdelivery but their performance was less than ideal in other criticalproperties. In particular, cubic liquid crystalline phases arerelatively viscous in nature. This makes application with a standardsyringe difficult, and possibly painful to the patient, and makessterilisation by filtration impossible because the composition cannot bepassed through the necessary fine-pored membrane.

WO2005/117830, for example, provides an improved system which has lowviscosity so as to improve the ease of manufacturing, handling andadministration with a standard syringe, allow for sterile filtration andreduce the pain on injection to the patient. However, for long-termdepot formulations and/or for formulations having protective or soothingproperties (such as surface-coating formulations for use in, forexample, per-oral applications), a crucial property is related to therobustness of the gel formed by the pre-formulation in the presence ofe.g. aqueous body fluids towards chemical and/or mechanical degradation,e.g. erosion/fragmentation/dissolution by endogenous surface activeagents (surfactants), lipid-degrading enzymes and/or physical break-up.

The present inventors have now established that providing apre-formulation comprising particular amphiphilic components, abiologically tolerable solvent and optionally at least one bioactiveagent, especially in a low viscosity phase such as molecular solution,gives a pre-formulation with greatly improved mechanical and/orchemical/enzymatic robustness. In addition, the pre-formulationmaintains many or all of the advantages of previous lipid depot systems,i.e. it is easy to manufacture, may be sterile-filtered, it has lowviscosity (allowing easy and less painful administration), allows a highlevel of bioactive agent to be incorporated (thus allowing a smalleramount of composition to be used) and/or forms a desired non-lamellardepot composition in vivo having a controllable “burst” or “non-burst”release profile. Advantages in terms of the protective and/or soothingnature of the compositions may also be maintained. The compositions arealso formed from materials that are non-toxic, biotolerable andbiodegradable.

Due to its improved resistance to degradation from erosion and/orfragmentation by physical and/or chemical means, the pre-formulation isespecially suitable for the formation of depot compositions followingparenteral administration for long-term drug delivery, e.g. several daysto several months after parenteral administration. The compositions arealso advantageous for non-parenteral (e.g. local or topical)administration to body cavities and/or surfaces of the body orelsewhere.

In particular, the compositions of the current invention are moreresistant to chemical/biological degradation and their mechanicalresistance is improved in comparison with existing lipid depot systems,while retaining the ability to spontaneously self-assemble in situ. Whentested in degradative/fragmenting systems which cause turbidity uponbreakup of the depot, the turbidity factor of the present formulationshas been demonstrated as being a factor of ten lower than for theprevious lipid based liquid crystal forming systems. This makes thecompositions of the invention particularly effective in terms of thelongevity of release. They are also well suited for application in areaswith high erosion/degradation problems, for example per-oralapplication, or lower-GI-tract applications.

A lipid-based, slow-release composition is described in WO2006/131730for GLP-1 and analogues thereof using lipid mixtures comprisingphosphatidyl choline. This is a highly effective formulation, but theconcentration of active agent which can be included in the formulationis limited by its solubility. Evidently, a higher concentration ofactive agent, together with improved mechanical and/orchemical/enzymatic robustness allows for the possibility of even longerduration depot products, products maintaining a higher systemicconcentration, and products having a smaller injection volume, all ofwhich factors are of considerable advantage under appropriatecircumstances. It would thus be of considerable value to establish a wayby which higher concentrations of active agents could be included in alipid-based depot formulation.

The present inventors have now further established that by incorporatingat least one polar solvent a pre-formulation may be generated addressingmany of the shortfalls of known depot formulations, and which may beapplied to provide an improved controlled release of peptide activeagent. By use of specific components in carefully selected ratios, andin particular with a mixture of an alcohol and a polar solvent, a robustdepot formulation can be generated having a combination of propertiesexceeding the performance of even the known lipid controlled-releasecompositions.

SUMMARY OF THE INVENTION

Viewed from a first aspect, the invention thus provides apre-formulation comprising a low viscosity, non-liquid crystalline,mixture of:

-   -   a. at least one diacyl glycerol and/or at least one tocopherol;    -   b. at least one phospholipid component comprising phospholipids        having        -   i. polar head groups comprising more than 50% phosphatidyl            ethanolamine, and        -   ii. two acyl chains each independently having 16 to 20            carbons wherein at least one acyl chain has at least one            unsaturation in the carbon chain, and there are no more than            four unsaturations over the two carbon chains;    -   c. at least one biocompatible, oxygen containing, low viscosity        organic solvent;        wherein optionally at least one bioactive agent is dissolved or        dispersed in the low viscosity mixture;        and wherein the pre-formulation forms, or is capable of forming,        at least one non-lamellar (e.g. non-lamellar liquid crystalline)        phase structure upon contact with an aqueous fluid.

Generally, the aqueous fluid will be a body fluid such as fluid from amucosal surface, tears, sweat, saliva, gastro-intestinal fluid,extra-vascular fluid, extracellular fluid, interstitial fluid or plasma,and the pre-formulation will form a liquid crystalline phase structurewhen contacted with a body surface, area or cavity (e.g. in vivo) uponcontact with the aqueous body fluid. The pre-formulation of theinvention may optionally contain a certain amount of water prior toadministration, but this will not be sufficient to lead to the formationof the necessary liquid crystalline phase.

Thus in one embodiment applicable to all aspects of the invention, thepre-formulation further comprises:

-   -   d. up to 20 wt. % of at least one polar solvent by weight of        components a)+b)+c)+d), preferably wherein said polar solvent        has a dielectric constant of at least 28 measured at 25° C.,        more preferably at least 30 measured at 25° C.

In a second aspect the invention provides a method of delivery of abioactive agent to a human or non-human animal (preferably mammalian)body, this method comprising administering a pre-formulation comprisinga non-liquid crystalline, low viscosity mixture of:

-   -   a. at least one diacyl glycerol and/or at least one tocopherol;    -   b. at least one phospholipid component comprising phospholipids        having        -   i. polar head groups comprising more than 50% phosphatidyl            ethanolamine, and        -   ii. two acyl chains each independently having 16 to 20            carbons wherein at least one acyl chain has at least one            unsaturation in the carbon chain, and there are no more than            four unsaturations over the two carbon chains;    -   c. at least one biocompatible, oxygen containing, low viscosity        organic solvent;        and at least one bioactive agent is dissolved or dispersed in        the low viscosity mixture, whereby to form at least one        non-lamellar liquid crystalline phase structure upon contact        with an aqueous fluid in vivo following administration.

The method of administration suitable for the above method of theinvention will be a method appropriate for the condition to be treatedand the bioactive agent used. A parenteral depot will thus be formed byparenteral (e.g. subcutaneous or intramuscular) administration while abioadhesive non-parenteral (e.g. topical) depot composition may beformed by administration to the surface of skin, mucous membranes and/ornails, to opthalmological, nasal, oral or internal surfaces or tocavities such as oral, nasal, rectal, vaginal or buccal cavities, theperiodontal pocket or cavities formed following extraction of a naturalor implanted structure or prior to insertion of an implant (e.g a joint,stent, cosmetic implant, tooth, tooth filling or other implant).

Viewed from a further aspect, the invention provides a method for thepreparation of a liquid crystalline composition comprising exposing apre-formulation comprising a non-liquid crystalline, low viscositymixture of:

-   -   a. at least one diacyl glycerol and/or at least one tocopherol;    -   b. at least one phospholipid component comprising phospholipids        having        -   i. polar head groups comprising more than 50% phosphatidyl            ethanolamine, and        -   ii. two acyl chains each independently having 16 to 20            carbons wherein at least one acyl chain has at least one            unsaturation in the carbon chain, and there are no more than            four unsaturations over the two carbon chains;    -   c. at least one biocompatible, oxygen containing, low viscosity        organic solvent;        and optionally at least one bioactive agent dissolved or        dispersed in the low viscosity mixture, to an aqueous fluid in        vivo.

The liquid crystalline composition formed in this method may bebioadhesive as described herein. A further aspect of the invention thusresides in the generation of a bioadhesive formulation by administrationof any of the formulation precursors (pre-formulations) indicated hereinto a body surface, such as any of those body surfaces indicated herein.The increased robustness of the composition of the invention makes itparticularly suitable for administration of active agents over anincreased duration. In addition the composition demonstrates improvederosion-resistance which further increases the duration ofadministration enabling e.g. once monthly or once every three months(once quarterly) injections. In particular, because the formulations ofthe present invention show most unusual and surprising resistance todegradation by digestive systems, such as bile acids, a further highlyadvantageous application of the present pre-formulations is in per-oraladministration where other depot type systems are unsuitable. Parenteraland per-oral methods of administration are thus most preferred for thiscomposition.

Viewed from yet another aspect, the invention provides a process for theformation of a pre-formulation suitable for the administration of abioactive agent to a (preferably mammalian) subject, said processcomprising forming a non-liquid crystalline, low viscosity mixture of

-   -   a. at least one diacyl glycerol and/or at least one tocopherol;    -   b. at least one phospholipid component comprising phospholipids        having        -   i. polar head groups comprising more than 50% phosphatidyl            ethanolamine, and        -   ii. two acyl chains each independently having 16 to 20            carbons wherein at least one acyl chain has at least one            unsaturation in the carbon chain, and there are no more than            four unsaturations over the two carbon chains;    -   c. at least one biocompatible, oxygen containing, low viscosity        organic solvent;        and dissolving or dispersing at least one bioactive agent in the        low viscosity mixture, or in at least one of components a, b or        c prior to forming the low viscosity mixture.

Methods for the formation of pre-formulations of the present invention(with or without bioactive agents) will preferably comprise the mixingof components a), b) and c), as these components are described herein.Such a mixing method may in one embodiment comprise the mixing ofcomponents a) and b) prior to the addition of component c).Alternatively or additionally, the mixing of components a), b) and c)may comprise heating of a mixture of these components to a temperatureabove 24° C. (e.g. 25 to 50° C.) for a suitable period (e.g. for 1 to 24hours). Such a method will preferably take place under conditions suchthat a clear homogeneous mixture of a single phase is generated.

Viewed from another aspect the invention further provides the use of anon-liquid crystalline, low viscosity mixture of:

-   -   a. at least one diacyl glycerol and/or at least one tocopherol;    -   b. at least one phospholipid component comprising phospholipids        having        -   i. polar head groups comprising more than 50% phosphatidyl            ethanolamine, and        -   ii. two acyl chains each independently having 16 to 20            carbons wherein at least one acyl chain has at least one            unsaturation in the carbon chain, and there are no more than            four unsaturations over the two carbon chains;    -   c. at least one biocompatible, oxygen containing, low viscosity        organic solvent;        wherein at least one bioactive agent is dissolved or dispersed        in the low viscosity mixture, in the manufacture of a        pre-formulation for use in the sustained administration of said        active agent, wherein said pre-formulation is capable of forming        at least one non-lamellar liquid crystalline phase structure        upon contact with an aqueous fluid.

Such a use may be in the manufacture of a medicament for use in thetreatment, prevention and/or palliation of any condition indicatedherein.

In yet a further aspect the invention provides a method of treatment orprophylaxis of a human or non-human (preferably mammalian) animalsubject comprising administration of a pre-formulation according to thefirst aspect of the invention.

In a corresponding aspect, the present invention provides for apre-formulation as described in any embodiment herein for use intherapy, such as for use in any of those therapies described herein.Thus the pre-formations may be used in the treatment, prevention and/orpalliation of any condition indicated herein.

The pre-formulations of the present invention are highly advantageous inthat they are stable to prolonged storage in their final “administrationready” form. As a result, they may readily be supplied foradministration either by health professionals or by patients or theircareers, who need not be fully trained health professionals and may nothave the experience or skills to make up preparations following complexreconstitution schemes/instructions.

In a yet further aspect, the present invention provides a disposableadministration device (which is also to include a device component)pre-loaded with one or more than one measured dose of a pre-formulationof the present invention. Such a device will, in one embodiment,typically contain a single dose ready for administration and willgenerally be sterile-packed such that the composition is stored withinthe device until administration. Such an embodiment is particularlysuited to the depot aspects of the invention and is very much suited tothe parenteral depot aspects. Suitable devices include cartridges,ampoules and particularly syringes and syringe barrels, either withintegral needles or with standard (e.g. luer) fittings adapted to take asuitable disposable needle. In an alternative embodiment, the device maycontain a plurality of doses or administrations (e.g. 2 to 100 doses oradministrations) of the pre-formulation. Such an embodiment isparticularly suited to aspects of the present invention where nobioactive agent is present and/or to aspects of the present inventionwhere non-parenteral (e.g. topical) formulations (especially bioadhesiveformulations) are generated.

In an additional aspect, the present invention thus provides adisposable administration device pre-loaded with at least one measureddose of a pre-formulation comprising a low viscosity mixture of:

-   -   a. at least one diacyl glycerol and/or at least one tocopherol;    -   b. at least one phospholipid component comprising phospholipids        having        -   i. polar head groups comprising more than 50% phosphatidyl            ethanolamine, and        -   ii. two acyl chains each independently having 16 to 20            carbons wherein at least one acyl chain has at least one            unsaturation in the carbon chain, and there are no more than            four unsaturations over the two carbon chains;    -   c. at least one biocompatible, oxygen containing, low viscosity        organic solvent;        where at least one bioactive agent is optionally dissolved or        dispersed in the low viscosity mixture, and wherein the        pre-formulation forms, or is capable of forming, at least one        non-lamellar liquid crystalline phase structure upon contact        with an aqueous fluid.

The pre-filled devices of the invention may also suitably be included inan administration kit, which kit also forms a further aspect of theinvention. In a still further aspect, the invention thus provides a kitfor the administration of at least one bioactive agent, said kitcontaining a measured dose of a pre-formulation of the invention andoptionally an administration device or component thereof. Preferably thedose will be held within the device or component, which will be suitablefor i.m. or preferably s.c. administration. The kits may includeadditional administration components such as needles, swabs, etc. andwill optionally and preferably contain instructions for administration.Such instructions will typically relate to administration by a route asdescribe herein and/or for the treatment of a disease indicated hereinabove.

In a yet further aspect, the invention thus additionally provides a kitfor the administration of at least one somatostatin receptor agonist,said kit containing a measured dose of a formulation comprising a lowviscosity mixture of:

-   -   a. at least one diacyl glycerol and/or at least one tocopherol;    -   b. at least one phospholipid component comprising phospholipids        having        -   i. polar head groups comprising more than 50% phosphatidyl            ethanolamine, and        -   ii. two acyl chains each independently having 16 to 20            carbons wherein at least one acyl chain has at least one            unsaturation in the carbon chain, and there are no more than            four unsaturations over the two carbon chains;    -   c. at least one biocompatible, oxygen containing, low viscosity        organic solvent;        wherein at least one bioactive agent is optionally dissolved or        dispersed in the low viscosity mixture, and wherein the        pre-formulation forms, or is capable of forming, at least one        non-lamellar liquid crystalline phase structure upon contact        with an aqueous fluid.

In one embodiment, applicable to all aspects of the invention, theactive agent, when present, excludes somatostatin receptor agonists, inother words the active agent does not comprise any somatostatin receptoragonist.

In a further embodiment, the active agent, when present, may excludecertain specific somatostatin receptor agonists, namely pasireotide,octreotide and/or salts and mixtures thereof. In this embodiment, theactive agent may comprise somatostatin receptor agonists with theexception of pasireotide, octreotide and/or salts and mixtures thereof.

DETAILED DESCRIPTION

As used herein, the term “low viscosity mixture” is used to indicate amixture which may be readily administered to a subject and in particularreadily administered by means of a standard syringe and needlearrangement. This may be indicated, for example by the ability to bedispensed from a 1 ml disposable syringe through a 22 awg (or a 23gauge) needle by manual pressure. In a particularly preferredembodiment, the low viscosity mixture should be a mixture capable ofpassing through a standard sterile filtration membrane such as a 0.22 μmsyringe filter. In other preferred embodiments, a similar functionaldefinition of a suitable viscosity can be defined as the viscosity of apre-formulation that can be sprayed using a compression pump orpressurized spray device using conventional spray equipment. A typicalrange of suitable viscosities would be, for example, 0.1 to 5000 mPas.The viscosity is preferably 1 to 1000 mPas, more preferably 1 to 800mPas, such as 50 to 750 mPas, and most preferably 50 to 500 mPas at 20°C.

It has been observed that by the addition of small amounts of lowviscosity solvent, as indicated herein, a very significant change inviscosity can be provided. For example, the addition of only 5% solventcan reduce viscosity 100-fold and addition of 10% may reduce theviscosity up to 10,000 fold. In order to achieve this non-linear,synergistic effect, in lowering viscosity it is important that a solventof appropriately low viscosity and suitable polarity be employed. Suchsolvents include those described herein infra.

The solvents employed in the pre-formulation of the invention must bebiocompatible. In particular, it is preferred if the solvents used arenon-halogenated, in particular, non-chlorinated solvents. Preferablyhalogenated solvents, especially chlorinated solvents are excluded fromthe pre-formulation of the invention. Thus, in one embodiment, thepre-formulations of all aspects of the invention do not contain anysignificant amount of halogenated solvent. Thus for example, the amountof halogenated solvent may be below 1 wt % (e.g. 0 to 1 wt %) of thetotal weight of pre-formulation. This will preferably be less than 0.5%,more preferably less than 0.1% and more preferably less than 0.01% byweight.

Where percentages or ratios are specified herein, these will be byweight unless otherwise specified or context requires otherwise.Generally the percentages will be relative to a specified set ofcomponents, such as % of the total weight of components a), b) and c).However, where no other basis is specified, percentages will be byweight of the total precursor formulation (pre-formulation).

Particularly preferred examples of low viscosity mixtures are molecularsolutions and/or isotropic phases such as L₂ and/or L₃ phases. Asdescribed above, the L₃ is a non-lamellar phase of interconnected sheetswhich has some phase structure but lacks the long-range order of aliquid crystalline phase. Unlike liquid crystalline phases, which aregenerally highly viscous, L₃ phases are of lower viscosity. Obviously,mixtures of L₃ phase and molecular solution and/or particles of L₃ phasesuspended in a bulk molecular solution of one or more components arealso suitable. The L₂ phase is the so-called “reversed micellar” phaseor microemulsion. Most preferred low viscosity mixtures are molecularsolutions, L₃ phases and mixtures thereof. L₂ phases are less preferred,except in the case of swollen L₂ phases as described below.

The present invention provides a pre-formulation comprising componentsa, b, c and optionally at least one bioactive agent as indicated herein.One of the considerable advantages of the pre-formulations of theinvention is that components a and b may be formulated in a wide rangeof proportions. In particular, it is possible to prepare and usepre-formulations of the present invention having a much greaterproportion of phospholipid component b) to diacyl glycerol and/ortocopherol without risking phase separation and/or unacceptably highviscosities in the pre-formulation. The weight ratios of components a:bmay thus be anything from 80:20 right up to 5:95. Preferred ratios wouldgenerally be from 80:20 to 20:80, for example from 70:30 to 30:70.Preferably the ratios are in the range from 40:60 to 60:40. Mostpreferably the ratios are in the range from 45:55 to 55:45, for example48:52 to 52:48, especially around 50:50.

In one preferred embodiment of the invention, there is a greaterproportion of component b than component a. That is, the weight ratioa:b is below 50:50, e.g. 50:50 to 5:95, preferably, 48:52 to 20:80 andmore preferably 45:55 to 30:70.

The amount of component c in the pre-formulations of the invention willbe at least sufficient to provide a low viscosity mixture (e.g. amolecular solution, see above) of components a, b and c and will beeasily determined for any particular combination of components bystandard methods. The phase behaviour itself may be analysed bytechniques such as visual observation in combination with polarizedlight microscopy, nuclear magnetic resonance, X-ray diffraction andcryo-transmission electron microscopy (cryo-TEM) to look for solutions,L₂ or L₃ phases, or liquid crystalline phases. Viscosity may be measureddirectly by standard means. As described above, an appropriate practicalviscosity is that which can effectively be syringed and particularlysterile filtered. This will be assessed easily as indicated herein. Themaximum amount of component c to be included will depend upon the exactapplication of the pre-formulation but generally the desired propertieswill be provided by any amount forming a low viscosity mixture (e.g. amolecular solution, see above) and/or a solution with sufficiently lowviscosity. Since the administration of unnecessarily large amounts ofsolvent to a subject is generally undesirable the amount of component cwill typically be limited to no more than ten times (e.g. three times)the minimum amount required to form a low viscosity mixture, preferablyno more than five times and most preferably no more than twice thisamount. The composition of the present invention may, however, contain agreater quantity of solvent than would be acceptable in an immediatedosage composition. This is because the process by which the activeagents are slowly released (e.g. formation of shells of liquidcrystalline phase as described herein) also serves to retard the passageof solvent from the composition. As a result, the solvent is releasedover some time (e.g. minutes or hours) rather than instantaneously andso can be better tolerated by the body.

As a general guide, the weight of component c will typically be around 2to 40% of the total weight of components a), b) and c), or of the totalweight of components a), b), c) and d) when component d) is present.This proportion is preferably (especially for injectable depots) 4 to30%, for example 5 to 25% by weight. More preferably component c) is inthe range 7 to 20%, for example 9 to 18% by weight. For non-parenteral(e.g. per-oral) depots component c) is preferably in the range 2 to 30%,for example 2 to 20%. More preferably component c) is in the range 2 to10% by weight.

In one embodiment applicable to all aspects of the invention, thepre-formulation further comprises component d) at least one polarsolvent, which will typically be present at up to 20% by weight ofcomponents a)+b)+c)+d). Preferably component d) will be greater than 1%by weight of the pre-formulation, for example 1-20 wt. %, particularly1.2-20 wt. %, especially 2-18 wt. %. More preferably component d) ispresent in the range 5-15 wt. %, especially 6-12 wt. %.

Where present, it is preferable that said polar solvent will have adielectric constant of at least 28 measured at 25° C., more preferablyat least 30 measured at 25° C. Preferred polar solvents include water,propylene glycol (PG) and N-Methyl-2-pyrrolidone.

In one alternative embodiment, the compositions may exclude a polarsolvent (i.e. may exclude all solvents with dielectric constant above 30at 20° C.) with the optional exception of NMP.

Component a)—Diacyl Glycerol/Tocopherol

Component “a” as indicated herein is a neutral lipid componentcomprising a polar “head” group and also non-polar “tail” groups.Generally the head and tail portions of the lipid will be joined by anester moiety but this attachment may be by means of an ether, an amide,a carbon-carbon bond or other attachment. Specifically in thepre-formulation of the invention, component a is a diacyl glycerol andhas two non-polar “tail” groups.

Mono-acyl (“lyso”) lipids are typically less well tolerated in vivo andwhere present will form a minor part of component a) (e.g. less than10%). Preferably, for parenteral compositions there will be less than10% mono-acyl lipids present as a proportion of component a). Fornon-parenteral (e.g. per-oral) compositions preferably there will beless than 20% mono-acyl lipids present as a proportion of component a).Examples of mono-acyl lipids include glycerol monooleate (GMO).

The two non-polar groups may have the same or a differing number ofcarbon atoms and may each independently be saturated or unsaturated.Examples of non-polar groups include C₁₆-C₂₀ alkyl and alkenyl groups,which are typically present as the esters of long chain carboxylicacids. These are often described by reference to the number of carbonatoms and the number of unsaturations in the carbon chain. Thus, CX:Zindicates a hydrocarbon chain having X carbon atoms and Z unsaturations.Examples particularly include palmitoyl (C16:0), phytanoyl (C16:0),palmitoleoyl (C16:1), stearoyl (C18:0), oleoyl (C18:1), elaidoyl(C18:1), linoleoyl (C18:2), linolenoyl (C18:3), and arachidonoyl (C20:4)groups. Thus, typical non-polar chains are based on the fatty acids ofnatural ester lipids, including palmitic, phytanic, palmitolic, stearic,oleic, elaidic, linoleic, linolenic or arachidonic acids, or thecorresponding alcohols. Preferable non-polar chains are C₁₆-C₂₀ (e.g.C₁₆ to C₁₈₎ groups, especially C₁₈ groups. It is most preferred if thenon-polar tail groups of component a) consists essentially ofunsaturated C18 groups. Especially preferred are C18:1 and C18:2 groups(and their mixtures), for example oleyl (C18:1), and/or linoleyl (C18:2)groups. Thus, dioleyl, dilinoleyl and/or oleyl/linoleyl diacyl glycerolsand all mixtures thereof are highly suitable.

The diacyl glycerol, when used as all or part of component “a”, may besynthetic or may be derived from a purified and/or chemically modifiednatural sources such as vegetable oils. Mixtures of any number of diacylglycerols may be used as component a. Most preferably this componentwill include at least a portion of glycerol dioleate (GDO). A highlypreferred example is DAG comprising at least 50%, preferably at least70% and even comprising substantially 100% GDO. Where the amount of GDOis above 50% or above 70%, much of the remainder (e.g. more than 50% ormore than 75% or the remainder) may be dilinoleyl glycerol and/or oleyllinoleyl glycerol.

An alternative or additional highly preferred class of compounds for useas all or part of component a) are tocopherols. As used herein, the term“a tocopherol” is used to indicate the non-ionic lipid tocopherol, oftenknown as vitamin E, and/or any suitable salts and/or analogues thereof.Suitable analogues will be those providing the phase-behaviour, lack oftoxicity, and phase change upon exposure to aqueous fluids, whichcharacterise the compositions of the present invention. Such analogueswill generally not form liquid crystalline phase structures as a purecompound in water. The most preferred of the tocopherols is tocopherolitself, having the structure below. Evidently, particularly where thisis purified from a natural source, there may be a small proportion ofnon-tocopherol “contaminant” but this will not be sufficient to alterthe advantageous phase-behaviour or lack of toxicity. Typically, atocopherol will contain no more than 10% of non-tocopherol-analoguecompounds, preferably no more than 5% and most preferably no more than2% by weight.

In a further advantageous embodiment of the invention, component a)comprises at least 50%, preferably at least 70% and more preferablyconsists essentially of tocopherols, in particular tocopherol as shownabove.

A preferred combination of constituents for component a) is a mixture ofat least one DAG with at least one tocopherol. Preferably the DAG willhave C16-C18 alkyl or alkenyl non-polar tail groups, for example oleyl,dioleyl and/or linoleyl groups. Such mixtures include 2:98 to 98:2 byweight tocopherol:GDO, e.g. 10:90 to 90:10 tocopherol:GDO and especially20:80 to 80:20 of these compounds. Similar mixtures of tocopherol withother DAGs are also suitable.

Component a) may be present in the range 20 to 80% by weight of thetotal weight of components a), b) and c), or of the total weight ofcomponents a), b), c) and d) when component d) is present. Preferablycomponent a) will independently be present in the range 25 to 65 wt. %,for example 30 to 55 wt. %. Most preferably component a) will be presentin the range 35 to 45 wt. %.

Component b)—Phospholipid Component

Component “b” in the present invention is at least one phospholipidcomponent comprising phospholipids having

-   -   i. polar head groups comprising more than 50% phosphatidyl        ethanolamine, and    -   ii. two acyl chains each independently having 16 to 20 carbons        wherein at least one acyl chain has at least one unsaturation in        the carbon chain, and there are no more than four unsaturations        over the two carbon chains.

As with component a), this component comprises a polar head group and atleast one non-polar tail group. The difference between components a) andb) lies principally in the polar group. The non-polar portions may thussuitably be derived from the fatty acids or corresponding alcoholsconsidered above for component a). The phospholipid component b)comprises phospholipids containing two acyl groups which may be the sameor different.

Preferred phospholipid polar “head” groups include phosphatidylcholine(PC), phosphatidylethanolamine (PE), sphingomyelin (SM),phosphatidylinositol (PI) and comprise at least 50% PE. The mostpreferred polar group is thus phosphatidylethanolamine (PE).Phospholipid component b) comprises at least one phospholipid havingpolar head groups comprising more than 50% PE, preferably at least 75%PE, for example at least 80% PE or at least 90% PE. Preferablyphospholipid component b) comprises at least one phospholipid havingpolar head groups consisting of essentially 100% phosphatidylethanolamine (e.g. greater than 90% PE or greater than 95% PE).

In one embodiment applicable to all aspects of the invention componentb) further comprises at least one phospholipid having

-   -   i. polar head groups comprising more than 90% phosphatidyl        choline, and    -   ii. two acyl chains each independently having 16 to 20 carbons        wherein at least one acyl chain has at least one unsaturation in        the carbon chain, and there are no more than four unsaturations        over the two carbon chains.

Preferably phospholipid component b) will comprise phospholipidsselected from phosphatidyl ethanolamines, and mixtures of phosphatidylethanolamines with at least one phospholipid selected from phosphatidylcholines, phosphatidyl inositols, and sphingomyelins. It is preferred ifphospholipid component b) comprises at least 50% PE, e.g. more than 50%PE, preferably at least 70% PE and most preferably at least 80% PE.Component b) may consist essentially of 100% PE (e.g. >95% PE).

A typical phospholipid component b) may comprise PE and PC in a ratio inthe range of 51:49 to 90:10, for example 70:30 to 80:20.

Preferably component b) comprises a maximum of 25% ofphosphatidylcholine (PC), for example 20% PC or in the range of 0 to 10%PC. Preferably component b) comprises a maximum of 25% ofphosphatidylinositol (PI), for example 0 to 10% PI. Preferably componentb) comprises a maximum of 25% of sphingomyelin, for example 0 to 10%sphingomyelin. Most preferably component b) comprises a maximum of 25%of the combined contributions of PC, PI and/or sphingomyelin, forexample 0 to 10%.

Most preferably, phospholipid component b) comprises dioleoylphosphatidyl ethanolamine (DOPE), Soy PE and/or Egg PE, or mixtures ofat least one of DOPE/Soy PE/Egg PE with at least one of dioleoylphosphatidyl choline (DOPC), Soy PC(SPC), and/or Egg PC (EPC).

The phospholipid portion may be derived from a natural source. Suitablesources of phospholipids include egg, heart (e.g. bovine), brain, liver(e.g. bovine), milk and plant sources including soybean. Particularlypreferred are Soy and Egg phospholipids, especially Soy PE and/or EggPE. Such sources may provide one or more constituents of component b,which may comprise any mixture of phospholipids. Preferably component b)comprises Soy PE and/or Egg PE.

Phospholipid component b) (as a whole) preferably forms a reversedhexagonal liquid crystalline phase at 37° C. in the presence of excessaqueous phase, for example excess water.

In a preferred embodiment component b) comprises DOPE and DOPC and/orSoy PC and/or Egg PC, preferably in a ratio in the range of 65:35 to90:10, such as 85:15, for example 70:30 to 80:20.

Since the pre-formulations of the invention are to be administered to asubject for the controlled release of an active agent, it is preferablethat the components a and b are biocompatible. In this regard, it ispreferable to use, for example, diacyl glycerol and phospholipids ratherthan mono-acyl (lyso) compounds. A notable exception to this istocopherol, as described above. Although having only one alkyl chain,this is not a “lyso” lipid in the convention sense. The nature oftocopherol as a well tolerated essential vitamin makes it highlybiocompatible.

It is furthermore most preferable that the lipids and phospholipids ofcomponents a and b are naturally occurring (whether they are derivedfrom a natural source or are of synthetic origin). Naturally occurringlipids tend to be tolerable both systemically and locally with lesseramounts of inflammation and reaction from the body of the subject. Notonly is this more comfortable for the subject but it may increase theresidence time of the resulting depot composition, especially forparenteral depots, since less immune system activity is recruited to theadministration site. In certain cases it may, however, be desirable toinclude a portion of a non-naturally-occurring lipid in components aand/or b. This might be, for example an “ether lipid” in which the headand tail groups are joined by an ether bond rather than an ester. Suchnon-naturally-occurring lipids may be used, for example, to alter therate of degradation of the resulting depot-composition by having agreater or lesser solubility or vulnerability to breakdown mechanismspresent at the site of active agent release. Although all proportionsfall within the scope of the present invention, generally, at least 50%of each of components a and b will be naturally occurring lipids. Thiswill preferably be at least 75% and may be up to substantially 100%.Particularly preferred are Soy and/or Egg derived lipids.

Two particularly preferred combinations of components a and b are GDOwith DOPE, and tocopherol with DOPE, especially in the region 20-80 wt.% GDO/tocopherol, 20-80 wt. % DOPE and 2-40 wt. % solvent (especiallyethanol and NMP or mixtures thereof). More preferred is the combination35-65 wt. % component a), 35-65 wt. % component b), and 2-30 wt. %component c), of the total weight of components a), b) and c) (and d)where present). In one embodiment, the solvent component c) does notcomprise PG or other polar solvents present in optional component d).This applies particularly when optional polar solvent component d) ispresent.

In addition to amphiphilic components a and b, the pre-formulations ofthe invention may also contain additional amphiphilic components atrelatively low levels. In one embodiment of the invention, thepre-formulation contains up to 10%, preferably up to 7% (by weight ofcomponents a) and b)) of a charged amphiphile, particularly an anionicamphiphile such as a fatty acid. Preferred fatty acids for this purposeinclude caproic, caprylic, capric, lauric, myristic, palmitic, phytanic,palmitolic, stearic, oleic, elaidic, linoleic, linolenic, arachidonic,behenic or lignoceric acids, or the corresponding alcohols. Preferablefatty acids are palmitic, stearic, oleic and linoleic acids,particularly oleic acid.

Component b) may be present in the range 20 to 80% by weight of thetotal weight of components a), b) and c). Preferably component b) willbe present in the range 25 to 65 wt. %, for example 30 to 55 wt. %. Mostpreferably component b) will present in the range 35 to 45 wt. % of thetotal weight of components a), b) and c), or of the total weight ofcomponents a), b), c) and d) when component d) is present.

Components a) and b) may independently be present in the range 20 to 80%by weight of the total weight of components a), b) and c), or of thetotal weight of components a), b), c) and d) when component d) ispresent. Preferably components a) and b) will independently be presentin the range 25 to 65 wt. %, for example 30 to 55 wt. %. Most preferablycomponents a) and b) will independently be present in the range 35 to 45wt. %.

Preferably the total of components a) and b) will be at least 30% byweight of components a), b) and c), more preferably at least 60% byweight of components a), b) and c), or of the total weight of componentsa), b), c) and d) when component d) is present.

The total of the lipid components, i.e. component a) and component b),will preferably be at least 30% by weight of the completepre-formulation, more preferably at least 50% by weight of the completepre-formulation. In one embodiment, the total of components a), b), c),optional component d) where present, and any optional active agent wherepresent will amount to at least 70 wt. % of the total composition. Thismay preferably be at least 80, more preferably at least 90 wt. % and inone embodiment the pre-formulation will consist essentially of thesecomponents. By “consists essentially of” as used herein is indicated anamount of at least 90%, preferably at least 95% by weight.

In a preferred embodiment, the pre-formulation may have at least 15% ofcomponent a) and/or at least 15% of component b) by weight of componentsa)+b)+c), or of the total weight of components a), b), c) and d) whencomponent d) is present.

Component c)—Solvent

Component “c” of the pre-formulations of the invention is an oxygencontaining organic solvent. Since the pre-formulation is to generate adepot composition following administration (e.g. in vivo), upon contactwith an aqueous fluid, it is desirable that this solvent be tolerable tothe subject and be capable of mixing with the aqueous fluid, and/ordiffusing or dissolving out of the pre-formulation into the aqueousfluid. Solvents having at least moderate water solubility are thuspreferred.

In a preferred version, the solvent is such that a relatively smalladdition to the composition comprising a and b, i.e. below 20% (by wt),or more preferably below 10%, give a large viscosity reductions of oneorder of magnitude or more. As described herein, the addition of 10%solvent can give a reduction of two, three or even four orders ofmagnitude in viscosity over the solvent-free composition, even if thatcomposition is a solution or L₂ phase containing no solvent, or anunsuitable solvent such as water (subject to the special case consideredbelow), or glycerol.

Typical solvents suitable for use as component c include at least onesolvent selected from alcohols, ketones, esters (including lactones),ethers, amides and sulphoxides.

Examples of suitable alcohols include ethanol and isopropanol. Monoolsare preferred to diols and polyols. Where diols or polyols are used,this is preferably in combination with an at least equal amount ofmonool or other preferred solvent. Examples of ketones include acetoneand propylene carbonate. Suitable ethers include diethylether,glycofurol, diethylene glycol monoethyl ether, dimethylisobarbide, andpolyethylene glycols. Suitable esters include ethyl acetate andisopropyl acetate and dimethyl sulphide is as suitable sulphide solvent.Suitable amides and sulphoxides include N-methylpyrrolidone (NMP),2-pyrrolidone, dimethylacetamide (DMA) and dimethylsulphoxide (DMSO),respectively. Less preferred solvents include dimethyl isosorbide,tetrahydrofurfuryl alcohol, diglyme and ethyl lactate.

Since the pre-formulations are to be administered to a living subject,it is necessary that the solvent component c is sufficientlybiocompatible. The degree of this biocompatibility will depend upon theapplication method and since component c may be any mixture of solvents,a certain amount of a solvent that would not be acceptable in largequantities may evidently be present. Overall, however, the solvent ormixture forming component c must not provoke unacceptable reactions fromthe subject upon administration. Generally such solvents will behydrocarbons or preferably oxygen containing hydrocarbons, bothoptionally with other substituents such as nitrogen containing groups.It is preferable that little or none of component c contains halogensubstituted hydrocarbons since these tend to have lowerbiocompatibility. Where a portion of halogenated solvent such asdichloromethane or chloroform is necessary, this proportion willgenerally be minimised. Where the depot composition is to be formednon-parenterally a greater range of solvents may evidently be used thanwhere the depot is to be parenteral.

Component c as used herein may be a single solvent or a mixture ofsuitable solvents but will generally be of low viscosity. This isimportant because one of the key aspects of the present invention isthat it provides pre-formulations that are of low viscosity and aprimary role of a suitable solvent is to reduce this viscosity. Thisreduction will be a combination of the effect of the lower viscosity ofthe solvent and the effect of the molecular interactions between solventand lipid composition. One observation of the present inventors is thatthe oxygen-containing solvents of low viscosity described herein havehighly advantageous and unexpected molecular interactions with the lipidparts of the composition, thereby providing a non-linear reduction inviscosity with the addition of a small volume of solvent.

The viscosity of the “low viscosity” solvent component c (single solventor mixture) should typically be no more than 18 mPas at 20° C. This ispreferably no more than 15 mPas, more preferably no more than 10 mPasand most preferably no more than 7 mPas at 20° C.

The solvent component c will generally be at least partially lost uponin vivo formation of the depot composition, or diluted by absorption ofwater from the surrounding air and/or tissue. It is preferable,therefore, that component c be at least to some extent water miscibleand/or dispersible and at least should not repel water to the extentthat water absorption is prevented. In this respect also, oxygencontaining solvents with relatively small numbers of carbon atoms (forexample up to 10 carbons, preferably up to 8 carbons) are preferred.Obviously, where more oxygens are present a solvent will tend to remainsoluble in water with a larger number of carbon atoms. The carbon toheteroatom (e.g. N, O, preferably oxygen) ratio will thus often bearound 1:1 to 6:1, preferably 2:1 to 4:1. Where a solvent with a ratiooutside one of these preferred ranges is used then this will preferablybe no more than 75%, preferably no more than 50%, in combination with apreferred solvent (such as ethanol). This may be used, for example todecrease the rate of evaporation of the solvent from the pre-formulationin order to control the rate of liquid crystalline depot formation.

Preferably, component c) is selected from alcohols, ketones, esters,ethers, amides, sulphoxides and mixtures thereof. More preferablycomponent c) is selected from monool alcohols, diols, triols, ethers,ketones and amides. Most preferred solvents for component c) areselected from the group consisting of low molecular weight PEGs (200-500Dalton), ethanol, NMP, or mixtures thereof. Especially preferred areethanol and NMP or mixtures thereof.

As mentioned above, as a general guide, the weight of component c willtypically be around 2 to 40% of the total weight of components a), b)and c), or of the total weight of components a), b), c) and d) whencomponent d) is present. This proportion is preferably (especially forinjectable depots) 4 to 30%, for example 5 to 25% by weight. Morepreferably component c) is in the range 7 to 20%, for example 9 to 18%by weight.

Optional Component d)—Polar Solvent

Although it has previously been suggested that lipid controlled-releasecompositions should be formulated substantially in the absence of water,in order to avoid the conversion to high-viscosity liquid crystallinephases, it has now been established that a small and carefullycontrolled amount of a polar solvent such as water can provideconsiderable benefits. In particular, the inclusion of this polarsolvent (preferably comprising water) allows further improvements incontrolling the initial release of active agent, allows higher stableloading of some peptide active agents, provides faster depot formationand/or provides further reduced discomfort upon injection. Any one ofthese factors potentially provides a significant improvement in thecontext of therapeutic drug delivery, patient health and/or patientcompliance.

The pre-formulations of the present invention can thus also contain apolar solvent, component d), in addition to component c). A suitableamount of the combined solvents, i.e. c)+d), will typically be greaterthan 1% by weight of the pre-formulation, for example 2-30 wt. %,particularly 2-25 wt. %, especially 5-20 wt. %. More preferablycomponent d) is present in the range 5-15%, especially 6-12%, by weightof the total composition. Component d) is preferably water, propyleneglycol or mixtures thereof. In one preferred aspect, thepre-formulations of the invention contain ethanol as component c) withwater and/or propylene glycol as component d).

In one embodiment the pre-formulation comprises at least 1.5% (e.g. atleast 4.5%) water as part of component d) (by weight of the totalcomposition) with the remainder being propylene glycol. At least 5%water with the balance of component d) being PG is preferred. Componentd) may comprise or consist of water.

In an alternative embodiment, component d) may comprise or consist ofpropylene glycol.

Polar solvents suitable as optional component d) typically may have adielectric constant of at least 28 when measured at 25° C., for exampleat least 30 when measured at 25° C. Highly suitable polar solventsinclude water, PG and NMP, as well as binary and ternary mixturesthereof.

Preferably, polar solvents suitable as optional component d) are notincluded as part of the main solvent component c). For example,component c) may exclude water, propylene glycol and/or mixturesthereof.

Preferably the total level of components c) and d) is not more than 35wt. %, preferably not more than 30 wt. %, preferably 10-30 wt. %, mostpreferably 12-25% by weight of components a)+b)+c)+d).

The ratio of components c) and d) will also have potential advantages inthe compositions of the invention. In particular, by inclusion of somepolar solvent which is miscible with the mono-alcohol component(especially water), the slight sensation that may be caused at theinjection site from the alcohol content can be substantially eliminated.Thus, in one embodiment, the weight ratio of components c):d) may be inthe range 30:70 to 70:30, more preferably 40:60 to 60:40. In oneembodiment, the amount of alcohol component c) by weight is no greaterthan the amount of polar solvent d). Ratios of c):d) ranging from 30:70to 50:50 are thus appropriate in such an embodiment. Approximately equalamounts of components c) and d) are highly appropriate.

In a preferred combination, component a) is GDO or tocopherol, componentb) is DOPE or a mixture of DOPE and PC, component c) is ethanol, NMP ormixtures thereof, and component d) is water, PG or mixtures thereof, inthe ranges 35-65 wt. % component a), 35-65 wt. % component b), 2-20 wt.% component c), and 5-15 wt. % component d).

A highly preferred combination for the pre-formulation is GDO, DOPE,ethanol, and water/propylene glycol or mixtures thereof. As indicatedabove, appropriate amounts of each component suitable for thecombination are those amounts indicated herein for the individualcomponents, in any combination.

Preferably, components a), b) and c) make up 80 to 95% by weight of thetotal composition and component d) makes up 10 to 20% by weight of thetotal composition.

Bioactive Agent

The pre-formulations of the present invention preferably contain one ormore bioactive agents (described equivalently as “active agents”herein). Active agents may be any compound having a desired biologicalor physiological effect, such as a peptide, protein, drug, antigen,nutrient, cosmetic, fragrance, flavouring, diagnostic, pharmaceutical,vitamin, or dietary agent and will be formulated at a level sufficientto provide an in vivo concentration at a functional level (includinglocal concentrations for topical compositions). Under some circumstancesone or more of components a, b and/or c may also be an active agent,although it is preferred that the active agent should not be one ofthese components. Most preferred active agents are pharmaceutical agentsincluding drugs, vaccines, and diagnostic agents.

Drug agents that may be delivered by the present invention include drugswhich act on cells and receptors, peripheral nerves, adrenergicreceptors, cholinergic receptors, the skeletal muscles, thecardiovascular system, smooth muscles, the blood circulation system,endocrine and hormone system, blood circulatory system, synoptic sites,neuroeffector junctional sites, the immunological system, thereproductive system, the skeletal system, autacoid system, thealimentary and excretory systems, the histamine system, and the centralnervous system.

Examples of drugs which may be delivered by the composition of thepresent invention include, but are not limited to, antibacterial agents,immune modulating agents, including immunostimulants andimmunosuppressants, anticancer and/or antiviral drugs such as nucleosideanalogues, paclitaxel and derivatives thereof, anti inflammatorydrugs/agents, such as non-steroidal anti inflammatory drugs andcorticosteroids, cardiovascular drugs including cholesterol lowering andblood-pressure lowing agents, analgesics, anti-emetics includinghistamine H1, NK1 and 5-HT₃ receptor antagonists, corticosteroids andcannabinoids, antipsychotics and antidepressants including serotoninuptake inhibitors, prostaglandins and derivatives, vaccines, and bonemodulators. Diagnostic agents include radionuclide labelled compoundsand contrast agents including X-ray, ultrasound and MRI contrastenhancing agents. Nutrients include vitamins, coenzymes, dietarysupplements etc.

Particularly suitable active agents include those which would normallyhave a short residence time in the body due to rapid breakdown orexcretion and those with poor oral bioavailability. These includepeptide, protein and nucleic acid based active agents, hormones andother naturally occurring agents in their native or modified forms. Byadministering such agents in the form of a depot composition formed fromthe pre-formulation of the present invention, the agents are provided ata sustained level for a length of time which may stretch to days, weeksor even several months in spite of having rapid clearance rates. Thisoffers obvious advantages in terms of stability and patient complianceover dosing multiple times each day for the same period. In onepreferred embodiment, the active agent thus has a biological half life(upon entry into the blood stream) of less than 1 day, preferably lessthan 12 hours and more preferably less than 6 hours. In some cases thismay be as low as 1-3 hours or less. Suitable agents are also those withpoor oral bioavailability relative to that achieved by injection, forwhere the active agent also or alternatively has a bioavailability ofbelow 20%%, or preferably below 2%, especially below 0.2%, and mostpreferably below 0.1% in oral formulations.

Peptide and protein based active agents include human and veterinarydrugs selected from the group consisting of adrenocorticotropic hormone(ACTH) and its fragments, angiotensin and its related peptides,antibodies and their fragments, antigens and their fragments, atrialnatriuretic peptides, bioadhesive peptides, bradykinins and theirrelated peptides, calcitonin peptides including calcitonin and amylinand their related peptides, vasoactive intestinal peptides (VIP)including growth hormone releasing hormone (GHRH), glucagon, andsecretin, opioid peptides including proopiomelanocortin (POMC) peptides,enkephalin pentapeptides, prodynorphin peptides and related peptides,pancreatic polypeptide-related peptides like neuropeptide (NPY), peptideYY (PYY), pancreatic polypeptide (PPY), cell surface receptor proteinfragments, chemotactic peptides, cyclosporins, cytokines, dynorphins andtheir related peptides, endorphins and P-lidotropin fragments,enkephalin and their related proteins, enzyme inhibitors,immunostimulating peptides and polyaminoacids, fibronectin fragments andtheir related peptides, gastrointestinal peptides,gonadotrophin-releasing hormone (GnRH) agonists and antagonist,glucagon-like peptides 1 and 2, growth hormone releasing peptides,immunostimulating peptides, insulins and insulin-like growth factors,interleukins, luthenizing hormone releasing hormones (LHRH) and theirrelated peptides (which are equivalent to GnRH agonists as describedbelow), melanocortin receptor agonists and antagonists, melanocytestimulating hormones and their related peptides, nuclear localizationsignal related peptides, neurotensins and their related peptides,neurotransmitter peptides, opioid peptides, oxytocins, vasopressins andtheir related peptides, parathyroid hormone and its fragments, proteinkinases and their related peptides, somatostatins and their relatedpeptides, substance P and its related peptides, transforming growthfactors (TGF) and their related peptides, tumor necrosis factorfragments, toxins and toxoids and functional peptides such as anticancerpeptides including angiostatins, antihypertension peptides, anti-bloodclotting peptides, and antimicrobial peptides; selected from the groupconsisting of proteins such as immunoglobulins, angiogenins, bonemorphogenic proteins, chemokines, colony stimulating factors (CSF),cytokines, growth factors, interferons (Type I and II), interleukins,leptins, leukaemia inhibitory factors, stem cell factors, transforminggrowth factors and tumor necrosis factors. An interesting class ofbioactive agents suitable for the invention are peptide hormones,including those of the: glycoprotein hormone family (the gonadotropins(LH, FSH, hCG), thyroid stimulating hormone (TSH); proopiomelanocortin(POMC) family, adrenocorticotropic hormone (ACTH); the posteriorpituitary hormones including vasopressin and oxytocin, the growthhormone family including growth hormone (GH), human chorionicsomatomammotropin (hCS), prolactin (PRL), the pancreatic polypeptidefamily including PP, PYY and NPY; melanin-concentrating hormone, (MCH);the orexins; gastrointestinal hormones and peptides including GLP-1 andGIP; ghrelin and obestatin; adipose tissue hormones and cytokinesincluding leptin, adiponectin, and resistin; natriuretic hormones;parathyroid hormone (PTH);

the calcitonin family with calcitonin and amylin; the pancreatichormones including insulin, glucagon and somatostatin. All syntheticpeptides designed to have similar receptor affinity spectrums as theabove mentioned peptides are also very suitable for the invention.

A further considerable advantage of the depot compositions of thepresent invention is that active agents are released gradually over longperiods without the need for repeated dosing. The compositions are thushighly suitable for situations where patient compliance is difficult,unreliable or where a level dosage is highly important, such asmood-altering actives, those actives with a narrow therapeutic window,and those administered to children or to people whose lifestyle isincompatible with a reliable dosing regime and for “lifestyle” activeswhere the inconvenience of repeated dosing might outweigh the benefit ofthe active. Particular classes of actives for which this aspect offers aparticular advantage include contraceptives, hormones includingcontraceptive hormones, and particularly hormones used in children suchas growth hormone, anti-addictive agents, and drugs used in treatment ofpoorly compliant populations, such as patients suffering fromschizophrenia, Alzheimer, or Parkinson's disease, anti-depressants andanticonvulsants

Cationic peptides are particularly suitable for use where a portion ofthe pre-formulation comprises an anionic amphiphile such as a fatty acidor anionic lipid, including phosphatidic acid, phosphatidylglycerol,phosphatidylserine. In this embodiment, preferred peptides includeoctreotide, lanreotide, calcitonin, oxytocin, interferon-beta and-gamma, interleukins 4, 5, 7 and 8 and other peptides having anisoelectric point above pH 7, especially above pH 8.

In one preferred aspect of the present invention, the composition of theinvention is such that a reversed micellar cubic (I₂) phase, or a mixedphase including I₂ phase is formed upon exposure to aqueous fluids and apolar active agent is included in the composition. Particularly suitablepolar active agents include peptide and protein actives, oligonucleotides, and small water soluble actives, including those listedabove. Of particular interest in this aspect are the peptide octreotideand other somatostatin related peptides, interferons alpha and beta,glucagon-like peptide 1 and glucagon-like peptide 2 receptor agonists,leuprorelin and other GnRH agonists, abarelix and other GnRHantagonists, zolendronate and ibandronate and other bisphosphonates.

Since all of the μ-opioid receptor agonists of choice for the treatmentof moderate-to-severe chronic pain (morphine, hydromorphone, fentanyl,methadone, oxycodone, and buprenorphine) have the same mechanism ofaction, their physiochemical and pharmacokinetic characteristics aremore critical in determining the appropriate route of administration andproduct formulation to be used. For example, the short eliminationhalf-life of opioids such as morphine, hydromorphone, and oxycodonerequire that these agents be administered frequently to achievearound-the-clock analgesia, which makes them excellent candidates forlong acting release formulations. Fentanyl and buprenorphine undergosignificant first-pass metabolism and lacks sufficient bioavailabilityafter oral administration. Together with their high potency, fentanyland buprenorphine are excellent candidates for the long acting injectiondepot formulation of the invention. Sufentanil, remifentanil,oxymorphone, dimorphone, dihydroetorphine, diacetylmorphine are otherpotent opioid receptor agonists suitable for the invention.

Buprenorphine is also used for maintenance treatment of opioid addictionas well as potentially also cocaine and amphetamine and met-amphetamineaddiction, where current sublingual buprenorphine formulations sufferfrom low bioavailability, high variability and limited effect duration,resulting in issues with unpredictable dose response and withdrawalsymptoms, particularly in mornings. These issues effectively addressedby using the injection depot formulation of the invention, as areproblems with misuse and misdirection where the need for high sublingualdoses are exploited by injection, where the effect is significantlyhigher for the same dose, thus facilitating misuse of the drug.Similarly, opioid antagonists can be used for treating addiction using aconvenient injection depot system as provided by the invention. Suitableopiate antagonists for use with the invention are naloxone, nalmefene,and naltrexone.

Antipsychotics, including risperidone, iloperidone, paliperidone,olanzapine, ziprazidone and aripiprazole are also highly suitable forthe invention in view of the potential for improved treatment complianceby patients, as well as by providing stable plasma levels over time.Similarly, the invention is useful in the treatment of dementia,Alzheimer's disease and Parkinson's disease, which adversely affectcognition. Suitable active ingredients include donepezil, rivastigmine,galantamine, and emantine, and pramipexol.

A particular advantage of the present invention when used in combinationwith protein/peptide active agents is that aggregation of the activeagent is suppressed. In one preferred embodiment, the present inventionthus provides a depot precursor and particularly a depot composition asdescribed herein comprising at least one peptide or protein active agentwherein no more than 5% of the active agent is in aggregated form.Preferably no more than 3% is aggregated and most preferably no morethan 2% (especially less than 2%) is in aggregated form. Thisstabilisation of non-aggregated protein is highly advantageous from thepoint of view of high effectiveness, low side effects and predictableabsorption profile. Furthermore, it is increasingly expected thatprotein/peptide therapeutics will have low levels of protein aggregationin order to secure regulatory approval.

Gonadotropin-releasing hormone agonists (GnRH agonists) are syntheticpeptides modelled after the hypothalamic neurohormone GnRH thatinteracts with the gonadotropin-releasing hormone receptor to elicit itsbiologic response, the release of the pituitary hormones folliclestimulating hormone (FSH) and luthenizing hormone (LH). GnRH agonistsare useful in treatment of cancers that are hormonally sensitive andwhere a hypogonadal state decreases the chances of a recurrence. Thusthey are commonly employed in the medical management of prostate cancerand have been used in patients with breast cancer. Other indicationareas include treatment of delaying puberty in individuals withprecocious puberty, management of female disorders that are dependent onestrogen productions. In addition, women with menorrhagia,endometriosis, adenomyosis, or uterine fibroids may receive GnRHagonists to suppress ovarian activity and induce a hypoestrogenic state.

Gonadotropin-releasing hormone receptor agonists (GnRH-RAs), such asleuprolide (or leuprorelin), goserelin, histrelin, triptorelin,buserelin, deslorelin, nafarelin and related peptides are used orindicated for the treatment of a variety of conditions where they aretypically administered over an extended period. GnRH-RAs form apreferred group of active agents for use in the present invention.

GnRH itself is a post-translationally modified decapeptide of structurepyro-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂ (GnRH-I). Two naturalvarients are also known, GNRH-II having 5-His, 7-Trp, 8-Tyrsubstitutions and GnRH_III having 7-Trp, 8-Leu. Several peptideanalogues with agonistic properties are known, most of which have the10-Gly-NH₂ replaced with N-Et-NH₂. Fertirelin has 10-Gly to N-Et-NH₂substitution only, while analogues having additional substitutions overGnRH-I include Leuprorelin (Leuprolide), (6-D-Leu), Buserelin(6-Ser(Bu^(t))), Histrelin (6-d-His(Imbzl)), Deslorelin (6-d-Trp).Another common nona-peptide agonist is Goserelin which is substitutedwith 6-Ser(Bu^(t)) and has 10-Gly-NH₂ replaced by AzaGly-NH₂. Narafelin(6-d-Nal) and Triptorelin (6-d-Trp) both retain the 10-Gly-NH₂ group.The structures of the two most common GnRH agonists (Leuprolide andGoserelin) are shown below as acetate salts.

Leuprolide: pyro-Glu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro- N-Et-NH₂ (acetate) Goserelin:pyro-Glu-His-Trp-Ser-Tyr-D-Ser(Bu^(t))-Leu-Arg- Pro-Azgly-NH₂ (acetate)

A small number of GnRH antagonists are also known, again based on theGnRH-I structure. These include Abarelix(D-Ala-D-Phe-D-Ala-Ser-Tyr-D-Asp-Leu-Lys(^(i)Pr)-Pro-D-Ala), Antarelix(D-Nal-D-Phe-D-Pal-Ser-Phe-D-Hcit-Leu-Lys(^(i)Pr)-Pro-D-Ala); Cetrorelix(D-Nal-D-Phe-D-Pal-Ser-Tyr-D-Cit-Leu-Arg-Pro-D-Ala), Ganirelix(D-Nal-D-Phe-D-Pal-Ser-Tyr-D-hArg-Leu-HArg-Pro-D-Ala), Itrelix(D-Nal-D-Phe-D-Pal-Ser-NicLys-D-NicLys-Leu-Lys(^(i)Pr)-Pro-D-Ala) andNal-Glu (D-Nal-D-Phe-D-Pal-Ser-D-Glu-D-Glu-Leu-Arg-Pro-D-Ala).

Administration of single doses of a GnRH agonist, such as leuprolide,stimulates pituitary release of gonadotropins (i.e., LH and FSH),resulting in increased serum LH and FSH concentrations and stimulationof ovarian and testicular steroidogenesis. Transient increases in serumtestosterone and dihydrotestosterone (DHT) in males and in serum estroneand estradiol concentrations in premenopausal females are observedduring initial therapy with single daily doses of the drug.

Although the effect of a potent GnRH agonist during short-term and/orintermittent therapy is stimulation of steroidogenesis, the principaleffect of the drug in animals and humans during long-term administrationis inhibition of gonadotropin secretion and suppression of ovarian andtesticular steroidogenesis. The exact mechanism(s) of action has notbeen fully elucidated, but continuous therapy with a GnRH agonistapparently produces a decrease in the number of pituitary GnRH and/ortesticular LH receptors, resulting in pituitary and/or testiculardesensitization, respectively. The drug does not appear to affectreceptor affinity for gonadotropins. Leuprolide's mechanism of actionmay also involve inhibition and/or induction of enzymes that controlsteroidogenesis. Other mechanisms of action may include secretion of anLH molecule with altered biologic activity or impairment of normalpulsatile patterns of LH and FSH secretion.

A number of serious medical indications are related to and/or affectedby the concentration of gonadal steroid hormones. These include certainneoplastic diseases, including cancers, especially of the breast andprostate, and benign prostatic hypertrophy; premature or delayed pubertyin adolescents; hirsuitism; alzheimer's disease; and certain conditionsrelating to the reproductive system, such as hypogonadism, anovulation,amenorrhea, oligospermia, endometriosis, leiomyomata (uterine fibroids),premenstrual syndrome, and polycystic ovarian disease. Control of thissystem is also important in in vitro fertilisation methods.

Although treatment with a GnRH agonist might be expected to exacerbateconditions affected by gonadal steroid hormone concentration, thedown-regulation effect discussed above results in the decrease of thesehormones to castrate level if therapy is continued for around 2 weeks orlonger. As a result, hormone-receptive tumours such as certain prostateand breast cancer, as well as precocious puberty and many of the otherconditions mentioned above can be improved or palliated by long-termGnRH agonist therapy.

The pre-formulations of the present invention contain one or more GnRHanalogues or other active (see above) (which are intended by anyreference to “active agents” herein). Since GnRH is a peptide hormone,typical GnRH analogues will be peptides, especially of 12 or fewer aminoacids. Preferably such peptides will be structurally related to GnRH I,II and/or III, and/or one or more of the known analogues, includingthose listed here. Peptides may contain only amino acids selected fromthose 20 α-amino acids indicated in the genetic code, or more preferablymay contain their isomers and other natural and non-natural amino acids,(generally α, β or γ amino acids) and their analogues and derivatives.Preferred amino acids include those listed above as constituents of theknown GnRH analogues.

Amino acid derivatives are especially useful at the termini of thepeptides, where the terminal amino or carboxylate group may besubstituted by or with any other functional group such as hydroxy,alkoxy, carboxy, ester, amide, thio, amido, amino, alkyl amino, di- ortri-alkyl amino, alkyl (by which is meant, herein throughout C₁-C₁₂alkyl, preferably C₁-C₆ alkyl e.g. methyl, ethyl, n-propyl, isopropyl,n-butyl, iso-, sec- or t-butyl etc.), aryl (e.g phenyl, benzyl, napthyletc) or other functional groups, preferably with at least one heteroatomand preferably having no more than 10 atoms in total, more preferably nomore than 6.

Particularly preferred GnRH analogues are constrained peptides of 6 to12 alpha-amino acids, of which particular examples include thoseindicated above, and particularly leuprolide and goserelin, of thesequences indicated above.

By “GnRH analogues”, as used herein is indicated any GnRH agonist orantagonist, preferably peptides, peptide derivatives or peptideanalogues. Peptide derived GnRH agonists are most preferred, such asthose indicated above and especially leuprolide or goserelin.

The GnRH analogue will generally be formulated as 0.02 to 12% by weightof the total formulation. Typical values will be 0.1 to 10%, preferably0.2 to 8% and more preferably 0.5 to 6%. A GnRH analogue content ofaround 1-5% is most preferable.

Doses of the GnRH analogue suitable for inclusion in the formulation,and thus the volume of formulation used will depend upon the releaserate (as controlled, for example by the solvent type and amount use) andrelease duration, as well as the desired therapeutic level, the activityof the specific agent, and the rate of clearance of the particularactive chosen. Typically an amount of 0.1 to 500 mg per dose would besuitable for providing a therapeutic level for between 7 and 180 days.This will preferably be 1 to 200 mg. For leuprolide or goserelin, thelevel will typically be around 1 to 120 mg (e.g. for a 30 to 180 dayduration). Preferably, the amount of leuprolide will be around 0.02 to 1mg per day between injections, for depots designed for release over 30days to 1 year, preferably 3 to 6 months. Evidently, the stability ofthe active and linearity of the release rate will mean that the loadingto duration may not be a linear relationship. A depot administered every30 days might have, for example 2 to 30 mg or a 90 day depot have 6 to90 mg of active, such as one of the GnRH analogues indicated herein.

Where the active agent comprises a 5HT₃ antagonist or second generation5HT₃ antagonist, this is preferably selected from odansetron,tropisetron, granisetron, dolasetron, palonosetron, alosetron,cilansetron and/or ramosetron or mixtures thereof. Doses of the 5HT₃antagonist suitable for inclusion in the formulation, and thus thevolume of formulation used will depend upon the release rate (ascontrolled, for example by the solvent type and amount use) and releaseduration, as well as the desired therapeutic level, the activity of thespecific agent, and the rate of clearance of the particular activechosen. Typically an amount of 1 to 500 mg per dose would be suitablefor providing a therapeutic level for between 5 and 90 days. This willpreferably be 1 to 300 mg. For granisetron, the level will typically bearound 10 to 180 mg (e.g. for a 3 to 60 day duration). Preferably, theamount of granisetron will be around 0.2 to 3 mg per day betweeninjections, for depots designed for release over 30 days to 1 year,preferably 3 to 6 months. Evidently, the stability of the active andlinearity of the release rate will mean that the loading to duration maynot be a linear relationship. A depot administered every 30 days mighthave, for example 2 to 30 mg or a 90 day depot have 6 to 90 mg ofactive.

Somatostatins (Growth Hormone Release Inhibiting Factors, SSTs) arenatural peptide hormones with a wide distribution in animals, acting asneurotransmitters in the central nervous system, and having diverseparacrine/autocrine regulatory effects on several tissues. Twobiologically active products are known in higher species, SST-14 andSST-28, a congener of SST-14 extended at the N-terminus.

SST-14 is a 14 residue cyclic peptide hormone having the sequenceAla-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys, where the twocysteine residues are connected by a disulphide bridge to generate atype II β-turn at the key binding sequence of Phe-Trp-Lys-Thr. Thebiological half-life of natural SST-14 is very short (1-3 minutes) andso it is not, in itself, a viable therapeutic in current formulations,but an increasing number of somatostatin receptor agonists are becomingavailable with higher activities and/or longer clearance times in vivo.

Somatostatin receptor agonists (SRAs), such as SST-14, SST-28,octreotide, lanreotide, vapreotide, pasireotide (SOM 230) and relatedpeptides, are used or indicated in the treatment of a variety ofconditions where they are typically administered over an extendedperiod. SRAs form a preferred group of active agents for use in thepresent invention.

Octreotide, for example, is the synthetic octapeptide with sequenceD-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol (2-7 disulphide bridge) and istypically administered as an acetate salt. This SST-14 derivativeretains the key Phe-(D)Trp-Lys-Thr β-turn required for in vivo SST-likeactivity but, in contrast to the natural hormone, has a terminalhalf-life of around 1.7 hours. Octreotide is used in treatment ofconditions including carcinoid tumours and acromegaly, and is typicallyadministered over a sustained period of weeks, or more commonly manymonths or years. Somatostatin receptor agonists are of particularinterest for the treatment of many different types of cancers since awide variety of tumours are found to express somatostatin receptors(SSTRs). There are five known types of SSTRs (SSTR1-SSTR5), showingequally high affinity for SST-14. The most investigated somatostatinreceptor agonists, including octreotide, show high selectivity for SSTR2and SSTR5; thus, octreotide is of particular interest for the treatmentof tumours expressing these types of receptors.

The most common “simple” formulation of Octreotide is “Sandostatin”®from Novartis. This is an aqueous solution for subcutaneous (s.c)injection, and a 100 μg dose reaches a peak concentration of 5.2 ng/mlat 0.4 hours post injection. The duration of action can be up to 12hours but s.c. dosing is generally carried out every 8 hours. Evidently,s.c. injection 3 times daily for periods of months or years is not anideal dosing regime.

Pasireotide is a multireceptor-targeted somatostatin analogue with highaffinity for somatostatin receptor subtypes sstr1,2,3 and sstr5 that hasbeen developed for the treatment of neuroendocrine diseases. Twoformulations of pasireotide have currently been developed: animmediate-release formulation for subcutaneous (sc) injection and along-acting-release (LAR) formulation. The structure of pasireotide isas follows:

Pasireotide was initially developed by Novartis Pharma as a treatmentfor Cushing's disease/syndrome and acromegaly, but has potentialapplicability in the treatment of several conditions for whichsomatostatin analogues such as octreotide are indicated, includingcarcinoid tumours.

Following a single subcutaneous dose of pasireotide, human plasma levelstypically peak quickly, at around 15 minutes to 1 hour after dosing,with an initial half-life of 2-3 hours following that peak. Althoughclearance half-life is greater for later phases of the decline, it isclear that the Cmax/Cave for such a delivery will be rather high.

Pasireotide LAR is a long acting formulation of pasireotide whichaddresses some of the above issues. However, this is a polymermicroparticle based system with the inherent limitations of such asystem, as are known in the art and described herein above.

Carcinoid tumours are intestinal tumour arising from specialised cellswith paracrine functions (APUD cells). The primary tumour is commonly inthe appendix, where it is clinically benign. Secondary, metastatic,intestinal carcinoid tumours secrete excessive amounts of vasoactivesubstances, including serotonin, bradykinin, histamine, prostaglandins,and polypeptide hormones. The clinical result is carcinoid syndrome (asyndrome of episodic cutaneous flushing, cyanosis, abdominal cramps, anddiarrhea in a patient with valvular heart disease and, less commonly,asthma and arthropathy). These tumours may grow anywhere in thegastrointestinal tract (and in the lungs) with approximately 90% in theappendix. The remainder occurs in the ileum, stomach, colon or rectum.Currently, treatment of carcinoid syndrome starts with i.v. bolusinjection followed by i.v. infusion. When sufficient effect on symptomshas been established, treatment with a depot formulation of octreotideformulated in ploy lactic-co-glycolic acid (PLGA) microspheres isstarted. However, during the first two weeks or more after injection ofthe depot, daily s.c. injections with octreotide are recommended tocompensate for the slow release from the PLGA spheres.

Certain of the pre-formulations of the present invention contain saltsof one or more somatostatin receptor agonists (which are preferredexamples of the peptide actives, which in turn are intended by anyreference to “active agents” herein). Since SST-14 is a peptide hormone,typical somatostatin receptor agonists will be peptides, especially of14 or fewer amino acids. Preferably such peptides will be structurallyconstrained such as by being cyclic and/or having at least oneintra-molecular crosslink. Amide, ester or particularly disulphidecrosslinks are highly suitable. Preferred constrained peptides willexhibit a type-2 β turn. Such a turn is present in the key region ofsomatostatin. Peptides may contain only amino acids selected from those20 α-amino acids indicated in the genetic code, or more preferably maycontain their isomers and other natural and non-natural amino acids,(generally α, β or γ, L- or D-amino acids) and their analogues andderivatives. The term “somatostatin receptor agonist” as used herein mayoptionally also encompass SST-14 and/or SST-28, since these are viablepeptide actives when formulated as salts in the very high performanceslow-release formulations described herein.

Amino acid derivatives and amino acids not normally used for proteinsynthesis are especially useful at the termini of the peptides, wherethe terminal amino or carboxylate group may be substituted by or withany other functional group such as hydroxy, alkoxy, ester, amide, thio,amino, alkyl amino, di- or tri-alkyl amino, alkyl (by which is meant,herein throughout C₁-C₁₈ alkyl, preferably C₁-C₈ alkyl e.g. methyl,ethyl, n-propyl, isopropyl, n-butyl, iso-, sec- or t-butyl etc.), aryl(e.g phenyl, benzyl, napthyl etc) or other functional groups, preferablywith at least one heteroatom and preferably having no more than 10 atomsin total, more preferably no more than 6.

Particularly preferred somatostatin receptor agonists are constrainedpeptides of 6 to 10 α-amino acids, of which particular examples includeoctreotide, lanreotide (of sequenceNH₂-(D)Naph-Cys-Tyr-(D)Trp-Lys-Val-Cys-Thr-CONH₂ and its cyclicderivative of sequence NH₂-(D)Naph-Cys-Tyr-(D)Phe-Lys-Val-Cys-Thr-CONH₂both having a Cys-Cys intramolecular disulphide crosslink), SOM 230 (seestructure above) and vapreotide. Most preferred are octreotide andpasireotide.

The somatostatin receptor agonist will generally be formulated as 0.1 to10% by weight of the total formulation. Typical values will be 0.5 to9%, preferably 1 to 8% and more preferably 1 to 7%. A somatostatinreceptor agonist content of 2-5% is most preferable.

Doses of the somatostatin receptor agonist suitable for inclusion in theformulation, and thus the volume of formulation used, will depend uponthe release rate (as controlled, for example by the solvent type andamount use) and release duration, as well as the desired therapeuticlevel, the activity and the rate of clearance of the particular activechosen. Typically an amount of 1 to 500 mg per dose would be suitablefor providing a therapeutic level for between 7 and 90 days. This willpreferably be 5 to 300 mg. For octreotide, the level will typically bearound 10 to 180 mg (e.g. for a 30 to 90 day duration). Preferably, theamount of octreotide will be around 0.2 to 3 mg per day betweeninjections. Thus a depot administered every 30 days would have 6 to 90mg or a 90 day depot have 18 to 270 mg of octreotide.

For Pasireotide, the dosage would typically be an amount of around 0.05to 40 mg per week of depot duration, preferably 0.1 to 20 mg per weekduration (e.g. 1 to 5 mg per week) for a duration of 1 to 24 weeks,preferably 2 to 16 (e.g. 3, 4, 8, 10 or 12) weeks. In an alternativeembodiment the pre-formulation may be formulated for dosing weekly (e.g.every 7±1 days). A total dose of 0.05 to 250 mg of Pasireotide per dosewould be suitable for providing a therapeutic level for between 7 and168 days. This will preferably be 0.1 to 200 mg, e.g. 0.2 to 150 mg, 0.1to 100 mg, 20 to 160 mg etc. Evidently, the stability of the active andeffects on the release rate will mean that the loading to duration maynot be a linear relationship. A depot administered every 30 days mighthave, for example 0.2 to 20 mg of Pasireotide, or a 90 day depot mighthave 30 to 60 mg of Pasireotide.

Where the salt of a peptide active agent, such as an SRA, is used in theformulations of the present invention, this will be a biologicallytolerable salt. Suitable salts include the acetate, pamoate, chloride orbromide salts. The chloride salt is most preferred.

The amount of bioactive agent to be formulated with the pre-formulationsof the present invention will depend upon the functional dose and theperiod during which the depot composition formed upon administration isto provide sustained release. Typically, the dose formulated for aparticular agent will be around the equivalent of the normal daily dosemultiplied by the number of days the formulation is to provide release.Evidently this amount will need to be tailored to take into account anyadverse effects of a large dose at the beginning of treatment and sothis will generally be the maximum dose used. The precise amountsuitable in any case will readily be determined by suitableexperimentation.

Preferably, the pre-formulation of the invention will comprise 0.1-10wt. % of said active agent by weight of components a)+b)+c) (and d)where present).

Preferably the active agent where present is selected from:

Interferons; GnRH agonists buserelin, deslorelin, goserelin,leuprorelin/leuprolide, naferelin and triptorelin; GnRH antagonists,e.g. cetrorelix, ganirelix, abarelix, degarelix; glucagon-like peptide-1(GLP-1) and analogues thereof, e.g. GLP-1(7-37), GLP-1(7-36) amide,liraglutide, exenatide, and lixisenatide (AVE0010); glucagon-likepeptide 2 agonists (GLP-2) and analogues thereof, e.g. GLP-2 andElsiglutide (ZP1846); DPPIV inhibitors; somatostatins SST-14 and SST-28and somatostatin receptor (SSTR) agonists, e.g. octreotide, lanreotide,vapreotide, pasireotide.

Other peptides suitable for the invention include: angiopeptin,angiotensin I, II, III, antileukinate, anti-inflammatory peptide 2,aprotinin, bradykinin, bombesin, calcitonin, calcitriol, cholecystokinin(CCK), colony-stimulating factor, corticotropin-releasing factor,C-Peptide, DDAVP, dermorphin-derived tetrapeptide (TAPS), dynorphin,endorphins, endostatin, endothelin, endothelin-1, enkephalins, epidermalgrowth factor, erythropoietin, fibroblast growth factor, folliclestimulating hormone, follistatin, follitropin, galanin, galanin-likepeptide, galectin-1, gastrin, gastrin-releasing peptide, G-CSF, ghrelin,glial-derived neurotrophic factor, GM-CSF, granulocytecolony-stimulating factor, growth hormone, growth hormone-releasingfactor, hepatocyte growth factor, insulin, insulin-like growth factors-Iand I, interferons, interleukins, leptin, leukemia inhibitory factor,melanocortin 1, 2, 3, 4, melanocyte-stimulating hormone metastin,monocyte chemotactic protein-1 (MCP-1), morphiceptin, NEP1-40,neuropeptide Y, neuropeptide W, orexin-A & orexin-B, oxytocinp21-Cip1/WAF-1, TAT fusion protein, parathyroid hormone, pigmentepithelium-derived growth factor (PEDF), peptide, peptide, proreninhandle region, peptide YY (3-36), platelet activating factor,platelet-derived growth factor, prorenin decapeptide, protegrin-1, PR39,prolactin, relaxin, secretin, substance P, tumor necrosis factor,urocortin, vascular endothelial growth factor, vasoactive intestinalpolypeptide, vasopressin.

Most preferably the active agent is at least one selected frombuprenorphine, octreotide, pasireotide, leuprolide and goserelin. Forexample, at least one selected from buprenorphine, leuprolide andgoserelin.

In one embodiment, applicable to all aspects of the invention, theactive agent excludes somatostatin receptor agonists, in other words theactive agent does not comprise any somatostatin receptor agonist.

In a further embodiment, the active agent, when present, may excludecertain specific somatostatin receptor agonists, namely pasireotide,octreotide and/or salts and mixtures thereof. In this embodiment, theactive agent may comprise somatostatin receptor agonists with theexception of pasireotide, octreotide and/or salts and mixtures thereof.

In one embodiment, applicable to all aspects of the invention, thefollowing pre-formulation, together with devices and kits containingsaid pre-formulation, processes for its formation and/or delivery, andthe use of said pre-formulation may be excluded:

-   -   a pre-formulation comprising a low viscosity, non-liquid        crystalline, mixture of:        -   a. 25-55 wt. % of at least one diacyl glycerol and/or at            least one tocopherol;        -   b. 25-55 wt. % of at least one phospholipid component            comprising phospholipids having            -   i. polar head groups comprising more than 50%                phosphatidyl ethanolamine, and            -   ii. two acyl chains each independently having 16 to 20                carbons wherein at least one acyl chain has at least one                unsaturation in the carbon chain, and there are no more                than four unsaturations over two carbon chains;        -   c. 5-25 wt. % of at least one biocompatible, oxygen            containing, low viscosity organic solvent;    -   wherein 0.1-10 wt. % of at least one peptide active agent        comprising at least one somatostatin receptor agonist is        dissolved or dispersed in the low viscosity mixture;    -   and wherein the pre-formulation forms, or is capable of forming,        at least one non-lamellar liquid crystalline phase structure        upon contact with an aqueous fluid.

The nature of the components of the pre-formulations of the presentinvention is that they are typically naturally occurring and highlybiocompatible. They thus cause little or no irritation upon contact witha body surface and may serve to form a soothing and/or barrier layer atsuch a surface. In such circumstances, an additional effect may beprovided by an “active” bioactive agent, such as any of those describedherein. However, a beneficial property my exist as a result of thephysical and/or biological effects of the pre-formulation and/or thelong-acting composition which forms upon administration.

Thus, in one embodiment, the optional bioactive agent may be absent fromany of the formulations described herein, where context allows.

Administration

As mentioned above, the pre-formulation of the invention may beadministered and the methods of the invention applied using a routeappropriate for the condition to be treated and the bioactive agentused. The term “parenteral” as used herein is given its establishedmeaning of “through the skin” rather than all “non-oral” routes. Thusparenteral primarily indicates administration by injection, infusion andsimilar techniques (such as needle-less injection). The term“non-parenteral” thus covers application routes other than through theskin. A parenteral depot will thus be formed by parenteral (e.g.injectable, such as by subcutaneous or intramuscular injection)administration while a non-parenteral (e.g. per-oral, topical) depotcomposition may be formed by administration to the surface of skin,mucous membranes and/or nails, to opthalmological, nasal, oral orinternal surfaces or to cavities such as nasal, rectal, vaginal orbuccal cavities, the periodontal pocket or cavities formed followingextraction of a natural or implanted structure or prior to insertion ofan implant (e.g a joint, stent, cosmetic implant, tooth, tooth fillingor other implant).

In one embodiment, the pre-formulations of the present invention willgenerally be administered parenterally. This administration willgenerally not be an intra-vascular method but will preferably besubcutaneous intracavitary or intramuscular. Typically theadministration will be by injection, which term is used herein toindicate any method in which the formulation is passed through the skin,such as by needle, catheter or needle-less injector.

In parenteral (especially subcutaneous (s.c.)) depot precursors,preferred active agents are those suitable for systemic administrationincluding antibacterials (including amicacin, monocycline anddoxycycline), local and systemic analgesics (including tramadol,fentanyl, morphine, hydromorphone, buprenorphine, methadone, oxycodone,codeine, asperine, acetaminophen), immunosuppressants (such asthalidomide, lenalidomide, sirolimus, deforolimus, everolimus,temsirolimus, Umirolimus, zotarolimus), NSAIDS (such as ibuprofene,naproxene, keteprofene, diclofenac, indomethansine, sulindac, tolmethin,salysylic acids such as salisylamide, diflunisal), Cox1 or Cox2inhibitors (such as celecoxib, rofecoxib, valdecoxib), oncology andendocrinology agents (including octreotide, lanreotide, buserelin,luprorelin, goserelin, triptorelin, avorelin, deslorein, abarelix,degarelix, fulvestrant, interferon alpha, interferon beta, darbepoetinalpha, epoetin alpha, beta, delta, cytarabine, docetaxel, andpaclitaxel), antiemetics (like granisetron, odansetron, palonsetron,aprepitant, fosaprepitant, netupitant, dexamethasone, in particular 5HT₃antagonists or second generation 5HT₃ antagonists, preferably selectedfrom odansetron, tropisetron, granisetron, dolasetron, palonosetron,alosetron, cilansetron and/or ramosetron or mixtures thereof),antipsychotics (like bromperidol, risperidone, olanzapine, iloperidone,paliperadone, pipotiazine and zuclopenthixol), antivirals,anticonvulsants (for instance tiagabine topiramate or gabapentin) ornicotine, hormones (such as testosterone, testosterone cypionate, andtestosterone undecanoate, medroxyprogesterone, estradiol) growthhormones (like human growth hormone), and growth factors (likegranulocyte macrophage colony-stimulating factor), anti diabetic agents(such as GLP_1(7-36) amide, GLP-1(7-37), liraglutide, exenatide,lixisenatide, and glucagon), acetylcholinesterase receptor inhibitors(such as neostigmine, physostigmine, and rivastigmine), and pramipexol.

In an alternative embodiment, the formulations of the present inventionmay form non-parenteral depots where the active agent is slowly releasedat a body surface. It is especially important in this embodiment thatthe pre-formulations of the invention and/or the liquid crystallinedepot compositions formed therefrom should preferably be bioadhesive.That is to say that the compositions should coat the surface to whichthey are applied and/or upon which they form as appropriate and shouldremain even when this surface is subject to a flow of air or liquidand/or rubbing. It is particularly preferable that the liquidcrystalline depot compositions formed should be stable to rinsing withwater. For example, a small volume of depot precursor may be applied toa body surface and be exposed to a flow of five hundred times its ownvolume of water per minute for 5 minutes. After this treatment, thecomposition can be considered bioadhesive if less than 50% of thebioactive agent has been lost. Preferably this level of loss will bematched when water equalling 1000 times and more preferably 10000 timesthe volume of the composition is flowed past per minute for five, orpreferably 10 minutes.

Although the non-parenteral depot compositions of the present inventionmay absorb some or all of the water needed to form a liquid crystallinephase structure from the biological surfaces with which they arecontacted, some additional water may also be absorbed from thesurrounding air. In particular, where a thin layer of high surface areais formed then the affinity of the composition for water may besufficient for it to form a liquid crystalline phase structure bycontact with the water in the air. The “aqueous fluid” are referred toherein is thus, at least partially, air containing some moisture in thisembodiment.

Non-parenteral depot compositions will typically be generated byapplying the pre-formulation topically to a body surface or to a naturalor artificially generated body cavity and/or to the surface of animplant. This application may be by direct application of liquid such asby spraying, dipping, rinsing, application from a pad or ball roller,intra-cavity injection (e.g to an open cavity with or without the use ofa needle), painting, dropping (especially into the eyes) and similarmethods. A highly effective method is aerosol or pump spraying andevidently this requires that the viscosity of the pre-formulation be aslow as possible and is thus highly suited to the compositions of theinvention. Non-parenteral depots may, however, be used to administersystemic agents e.g. transmucosally or transdermally.

Non-parenteral depots may also be used for application to surfaces,particularly of implants and materials which will be in contact with thebody or a body part or fluid. Devices such as implants, catheters etc.may thus be treated e.g. by dipping or spraying with thepre-formulations of the invention, which will form a robust layer toreduce the introduction of infection. Anti-infective actives areparticularly suited to this aspect.

Conditions particularly suitable for causative or symptomatic treatmentby topical bioadhesive depot compositions of the present inventioninclude skin conditions (such as soreness resulting from any causeincluding chapping, scratching and skin conditions including eczema andherpes) eye conditions, genital soreness (including that due to genitalinfection such as genital herpes), infections and conditions for thefinger and/or toe nails (such as bacterial or fungal infections of thenails such as onychomycosis or poronychia). Topical-type bioadhesiveformulations may also be used to administer systemic active agents (e.g.medication), particularly by skin adsorption, oral, transdermal orrectal routes. Antiemetics and travel sickness medication is a preferredexample, as is nicotine (e.g. in anti-smoking aids). Where contextpermits, “topical application” as referred to herein includes systemicagents applied non-parenterally to a specific region of the body.

Periodontal infections are particularly suitable for treatment by thecompositions of the present invention. In particular, known compositionsfor treating periodontal infection are difficult to apply or aregenerally ineffective. The most widely used periodontal depotcomposition comprises insertion of a collagen “chip” into theperiodontal space, from which an anti-infective agent is released. Thischip is difficult to insert and does not form to match the shape andvolume of the periodontal space, so that pockets of infection may remainuntreated. In contrast to this, the compositions of the presentinvention, applied as a low viscosity pre-formulation, can be easily andquickly injected into the periodontal space and will flow to conformexactly to that space and fill the available volume. The compositionsthen quickly absorb water to form a robust gel which is resistant toaqueous conditions of the mouth. The only known previous attempt at suchan injectable periodontal treatment relied on dispersions of relativelyhigh viscosity which were difficult to apply and were subject toundesirable phase separation. All of these drawbacks are now addressedin the compositions of the present invention as described herein, whichfurthermore can be made more strong and durable than previouslydescribed lipid liquid crystalline systems. The current inventionprovides compositions which are highly robust, thus being especiallysuitable for use in the aqueous conditions found in the mouth.

Non-parenteral depot compositions are also of significant benefit incombination with non-pharmaceutical active agents, such as cosmeticactives, fragrances, essential oils etc. Such non-pharmaceutical depotswill maintain the important aspects of bioadhesion and sustained releaseto provide prolonged cosmetic effects, but may easily be applied byspraying or wiping.

Active agents particularly suited to non-parenteral (e.g. oral ortopical) depot administration, which comprises intra oral, buccal,nasal, ophthalmic, dermal, vaginal delivery routes, includeantibacterials such as chlorhexidine (e.g. chlorhexidine digluconate orchlorhexidine dihydrochloride), chloramphenicol, triclosan,tetracycline, terbinafine, tobramycin, fusidate sodium, butenafine,metronidazole (the latter particularly for the (e.g. symptomatic)treatment of acne rosacea—adult acne or some vaginal infections),antiviral, including acyclovir, anti infectives such as bibrocathol,ciprofloxacin, levofloxacin, local analgesics such as benzydamine,lidocaine, prilocalne, xylocalne, bupivacaine, analgesics such astramadol, fentanyl, morphine, hydromorphone, methadone, oxycodone,codeine, asperine, acetaminophen, antiemetics (like granisetron,odansetron, palonsetron, aprepitant, fosaprepitant, netupitant,dexamethasone, in particular 5HT₃ antagonists or second generation 5HT₃antagonists, preferably selected from odansetron, tropisetron,granisetron, dolasetron, palonosetron, alosetron, cilansetron and/orramosetron or mixtures thereof), NSAIDS such as ibuprofen, flurbiprofen,naproxene, ketoprofen, ketorolac, fenoprofen, diclofenac, etodalac,diflunisal, oxaproxin, piroxicam, piroxicam, indomethansine, sulindac,tolmethin, salysylic acids such as salisylamide and diflunisal, Cox1 orCox2 inhibitors such as celecoxib, rofecoxib or valdecoxib,corticosteroids, anticancer and immuno stimulating agents (for instance,methylaminolevulinat hydrocloride, interferon alpha and beta),anticonvulsants (for instance tiagabine topiramate or gabapentin),hormones (such as testosterone, and testosterone undecanoate,medroxyprogesterone, estradiol) growth hormones (like human growthhormone), and growth factors (like granulocyte macrophagecolony-stimulating factor), immuno suppressants (cyclosporine,sirolimus, tacrolimus, everolimus), nicotine and antivirals (e.g.acyclovir).

Phase Structures

The pre-formulations of the present invention provide non-lamellarliquid crystalline depot compositions upon exposure to aqueous fluids,especially in vivo and in contact with body surfaces. In a preferredembodiment the liquid crystalline phases of the invention are formed insitu.

As used herein, the term “non-lamellar” is used to indicate a normal orreversed liquid crystalline phase (such as a cubic or hexagonal phase)or the L3 phase or any combination thereof. The term liquid crystallineindicates all hexagonal, all cubic liquid crystalline phases and/or allmixtures thereof. Hexagonal as used herein indicates “normal” or“reversed” hexagonal (preferably reversed) and “cubic” indicates anycubic liquid crystalline phase, preferably reversed. By use of thepre-formulations of the present invention it is possible to generate anyphase structure present in the phase-diagram of components a and b withwater. This is because the pre-formulations can be generated with awider range of relative component concentrations than previous lipiddepot systems without risking phase separation or resulting in highlyviscous solutions for injection. In particular, the present inventionprovides for the use of phospholipid concentrations above 50% relativeto the total amphiphile content. This allows access to phases only seenat high phospholipid concentrations, particularly the hexagonal liquidcrystalline phases.

Preferably in the pre-formulation of the invention the liquidcrystalline phase structure formed upon contact with an aqueous fluid isa reversed hexagonal phase structure (H₂) and/or a reversed cubic phasestructure (I₂) or a mixture or intermediates thereof. With intermediateswe refer to phases with mean curvatures between the mean curvature of H₂and I₂ phases, respectively, and which position in a phase diagram isbetween these two phases in case both are present. Preferably the liquidcrystalline phase structure is selected from H₂, I₂ or mixtures thereof.

For many combinations of lipids, only certain non-lamellar phases exist,or exist in any stable state. It is a surprising feature of the presentinvention that compositions as described herein frequently exhibitnon-lamellar phases which are not present with many other combinationsof components. In one particularly advantageous embodiment, therefore,the present invention relates to compositions having a combination ofcomponents for which an I₂ and/or L₂ phase region exists when dilutedwith aqueous solvent. The presence or absence of such regions can betested easily for any particular combination by simple dilution of thecomposition with aqueous solvent and study of the resulting phasestructures by the methods described herein.

In a highly advantageous embodiment, the compositions of the inventionmay form an I₂ phase, or a mixed phase including I₂ phase upon contactwith water. The I₂ phase is a reversed cubic liquid crystalline phasehaving discontinuous aqueous regions. This phase is of particularadvantage in the controlled release of active agents and especially incombination with polar active agents, such as water soluble activesbecause the discontinuous polar domains prevent rapid diffusion of theactives. Depot precursors in the L₂ are highly effective in combinationwith an I₂ phase depot formation. This is because the L₂ phase is aso-called “reversed micellar” phase having a continuous hydrophobicregion surrounding discrete polar cores. L₂ thus has similar advantageswith hydrophilic actives.

In transient stages after contact with body fluid the composition cancomprise multiple phases since the formation of an initial surface phasewill retard the passage of solvent into the core of the depot,especially with substantial sized administrations of internal depots.Without being bound by theory, it is believed that this transientformation of a surface phase, especially a liquid crystalline surfacephase, serves to dramatically reduce the “burst/lag” profile of thepresent compositions by immediately restricting the rate of exchangebetween the composition and the surroundings. Transient phases mayinclude (generally in order from the outside towards the centre of thedepot): H₂ or L_(α), I₂, L₂, and liquid (solution). It is highlypreferred that the composition of the invention is capable forming atleast two and more preferably at least three of these phasessimultaneously at transient stages after contact with water atphysiological temperatures. In particular, it is highly preferred thatone of the phases formed, at least transiently, is the I₂ phase.

It is important to appreciate that the pre-formulations of the presentinvention are of low viscosity. As a result, these pre-formulations mustnot be in any bulk liquid crystalline phase since all liquid crystallinephases have a viscosity significantly higher than could be administeredby syringe or spray dispenser. The pre-formulations of the presentinvention will thus be in a non-liquid crystalline state, such as asolution, L₂ or L₃ phase, particularly solution or L₂. The L₂ phase asused herein throughout is preferably a “swollen” L₂ phase containinggreater than or about 10 wt % of solvent (component c) having aviscosity reducing effect. This is in contrast to a “concentrated” or“unswollen” L₂ phase containing no solvent, or a lesser amount ofsolvent, or containing a solvent (or mixture) which does not provide thedecrease in viscosity associated with the oxygen-containing, lowviscosity solvents specified herein.

Upon administration, the pre-formulations of the present inventionundergo a phase structure transition from a low viscosity mixture to ahigh viscosity (generally tissue adherent) depot composition. Generallythis will be a transition from a molecular mixture, swollen L₂ and/or L₃phase to one or more (high viscosity) liquid crystalline phases such asnormal or reversed hexagonal or cubic liquid crystalline phases ormixtures thereof. As indicated above, further phase transitions may alsotake place following administration. Obviously, complete phasetransition is not necessary for the functioning of the invention but atleast a surface layer of the administered mixture will form a liquidcrystalline structure. Generally this transition will be rapid for atleast the surface region of the administered formulation (that part indirect contact with air, body surfaces and/or body fluids). This willmost preferably be over a few seconds or minutes (e.g. up to 30 minutes,preferably up to 10 minutes, more preferably 5 minutes of less). Theremainder of the composition may change phase to a liquid crystallinephase more slowly by diffusion and/or as the surface region disperses.

In one preferred embodiment, the present invention thus provides apre-formulation as described herein of which at least a portion forms ahexagonal liquid crystalline phase upon contact with an aqueous fluid.The thus-formed hexagonal phase may gradually disperse, releasing theactive agent, or may subsequently convert to a cubic liquid crystallinephase, which in turn then gradually disperses. It is believed that thehexagonal phase will provide a more rapid release of active agent, inparticular of hydrophilic active agent, than the cubic phase structure,especially the I₂ and L₂ phase. Thus, where the hexagonal phase formsprior to the cubic phase, this will result in an initial release ofactive agent to bring the concentration up to an effective levelrapidly, followed by the gradual release of a “maintenance dose” as thecubic phase degrades. In this way, the release profile may becontrolled.

Without being bound by theory, it is believed that upon exposure (e.g.to body fluids), the pre-formulations of the invention lose some or allof the organic solvent included therein (e.g. by diffusion and/orevaporation) and take in aqueous fluid from the bodily environment (e.g.moist air close to the body or the in vivo environment) such that atleast a part of the formulation generates a non-lamellar, particularlyliquid crystalline phase structure. In most cases these non-lamellarstructures are highly viscous and are not easily dissolved or dispersedinto the in vivo environment and are bioadhesive and thus not easilyrinsed or washed away. Furthermore, because the non-lamellar structurehas large polar, apolar and boundary regions, it is highly effective insolubilising and stabilising many types of active agents and protectingthese from degradation mechanisms. As the depot composition formed fromthe pre-formulation gradually degrades over a period of days, weeks ormonths, the active agent is gradually released and/or diffuses out fromthe composition. Since the environment within the depot composition isrelatively protected, the pre-formulations of the invention are highlysuitable for active agents with a relatively low biological half-life(see above).

Robustness

The pre-formulations of the invention have improved robustness incomparison with liquid depot formulations known in the art. This isdemonstrated by their improved performance in terms oferosion/fragmentation and mechanical/degradation robustness.

A way to study the robustness in vitro is to simulate in vivo conditionsby subjecting the lipid gels to a surfactant-rich aqueous environmentand subsequently measuring the increased turbidity (or apparentabsorbance) of the aqueous phase resulting from surfactant-eroded lipidfragments. Such lipid fragments are released into the solution assuspended particles and give rise to substantial increase in solutionturbidity due to light scattering. Bile salts are often used as thesurfactant of choice for studying formulation dissolution given theirbiological relevance and endogenous nature. They are also among the mostchallenging constituents of the in-vivo environment for a depot systemto tolerate and so a system which is resistant to bile salts ispotentially of considerably value in drug delivery.

The turbidity factor of the pre-formulations of the invention wasmeasured using the process described in example 3. The turbidity factormay be considered a measure of the robustness of the pre-formulation inrespect of erosion/fragmentation, i.e. chemical degradation. Theturbidity factor (TF) is thus defined herein as the absorbance (orturbidity) at 600 nm of the aqueous phase resulting from placing a 200mg aliquot of pre-formulation in 5 ml of a 0.1 wt. % solution of sodiumtaurocholate in phosphate buffered saline (pH 7.4), at 37° C. for 6hours under 150 rpm rotation.

The pre-formulations of the invention have a reduced turbidity factor incomparison with that of existing formulations. Preferably the turbidityfactor is decreased by at least 50% in comparison with existingpre-formulations. More preferably the turbidity factor of thepre-formulations of the invention is decreased by at least 60% incomparison with existing pre-formulations. For example the turbidityfactor of the invention may be equal to or less than half, preferablyless than 40% of the turbidity factor of the existing pre-formulation.

It is a considerable and surprising benefit of the present precursorformulations that they show markedly superior resistance to degradationin comparison with corresponding formulations in which the phospholipidcomponent (component b)) is phosphatidyl choline. Thus, for example, theturbidity factor over an equivalent composition in which component b) isPC is decreased by at least 50%. More preferably the turbidity factor ofthe pre-formulations of the invention are decreased by at least 60% incomparison with equivalent pre-formulations in which component b) is PC(e.g. soy PC). For example the turbidity factor of the invention may beequal to or less than half, preferably less than 40% of the turbidityfactor of the corresponding PC-containing pre-formulation.

Preferably the turbidity factor of the pre-formulations according to theinvention may be approximately 0.6 or less, for example, 0.4. Morepreferably the turbidity factor may be 0.3 or less, for example 0.25 orless. Most preferably the turbidity factor may be 0.2 or less.

In comparison with existing liquid depot pre-formulations (such as thosein which component b) is PC, such as soy PC), preferably the turbidityfactor of the pre-formulations of the invention is reduced by at least afactor of three, for example a factor of five, more preferably a factoror eight and most preferably a factor of ten.

In a preferred embodiment, the absorbance value of a PE-basedpre-formulation measured according to example 3 will be in the range ofone third to one eighth of the corresponding PC-based formulation. Forexample, a GDO/PE based pre-formulation may have an absorbance value ofone third to one eighth of the corresponding GDO/PC composition.

It is a particular and unexpected advantage of the presentpre-formulations that they show remarkable resistance to bile aciddegradation. This has considerable advantages in providing compositionsthat may be administered orally and will persist through the digestivetract for some time without being broken down/digested. In particular,the precursor formulations of the present invention are useful for thedelivery of active agents to the GI tract. Since the compositionfurthermore protects the entrained active agent from the conditions ofthe GI tract, this embodiment may be applied in combination with activesthat are susceptible to breakdown in the GI tract, such as peptides.Many peptides are described herein and they may be used appropriately inthis embodiment. Delivery of an active agent to a portion of the GItract below the bile duct is a highly preferred embodiment that may beapplied to all appropriate aspects of the invention. Thepre-formulations may thus be for delivery of an active agent to the GItract below the bile duct, etc. Methods of treatment and similarapplications may correspondingly be for treatment of a condition in aregion of the GI tract below the bile duct.

In combination with the features and preferred features indicatedherein, the pre-formulations of the invention may have one or more ofthe following preferred features independently or in combination:

The optional active agent is present in the pre-formulation;

The pre-formulation forms a liquid crystalline phase structure which isbioadhesive;

Preferably said liquid crystalline phase structure is a reversedhexagonal phase structure or a reversed cubic phase structure ormixtures thereof, such as H₂ and/or I₂ or mixtures thereof;

The non-polar tail groups of component a) each independently consistessentially of unsaturated C18 groups; or component a) consistsessentially of at least one tocopherol; or component a) consistsessentially of a mixture of glycerol dioleate (GDO) and tocopherol;

Component b) is selected from phosphatidyl ethanolamines, or mixtures ofphosphatidyl ethanolamines with at least one selected from phosphatidylcholines, phosphatidyl inositols and sphingomyelins;

The phospholipid component b) comprises at least 50% PE, preferably atleast 75% PE and most preferably essentially 100% PE;

The phospholipid component b) comprises 10-49% PC, for example 20% PC;

The phospholipid component b) comprises a phospholipid having polar headgroups consisting of essentially 100% phosphatidyl ethanolamine;

The phospholipid component b) further comprises a phospholipid havingpolar head groups consisting of greater than 90% phosphatidyl choline(e.g. at up to 49% of component b));

The pre-formulation has a viscosity in the range of 0.1 to 5000 mPas;

The pre-formulation has a molecular solution, L₂ and/or L₃ phasestructure;

The pre-formulation has a ratio of a) to b) of between 80:20 and 5:95 byweight;

The pre-formulation has at least 15% of component a) and/or at least 15%of component b) by weight of components a)+b)+c);

The pre-formulation has 2 to 40% component c) by weight of componentsa)+b)+c);

Component c) is selected from alcohols, ketones, esters, ethers, amides,sulphoxides and mixtures thereof;

The pre-formulation further comprises component d) up to 20 wt. % of atleast one polar solvent by weight of components a)+b)+c)+d);

The polar solvent has a dielectric constant of at least 28 measured at25° C., preferably at least 30 measured at 25° C.;

Component d) is selected from water, propylene glycol, NMP and mixturesthereof; Component d) comprises at least 2% water;

The pre-formulation additionally comprises up to 10% by weight of a)+b)of a charged amphiphile;

The pre-formulation has 0.1-10 wt. % of said active agent by weight ofcomponents a)+b)+c)+d);

The active agent is selected from drugs, antigens, nutrients, cosmetics,fragrances, flavourings, diagnostic agents, vitamins, dietarysupplements and mixtures thereof;

Where said active agent is a drug, said drug is selected fromhydrophilic small molecule drugs, lipophilic small molecule drugs,amphiphilic small molecule drugs, peptides, proteins, oligonucleotidesand mixtures thereof;

Said drug is selected from buprenorphine, fentanyl, granisetron,odansetron, palonsetron, aprepitant, fosaprepitant, netupitant,dexamethasone somatostatin related peptides, somatostatin 14,somatostatin 28, octreotide, lanreotide, vapreotide, pasireotide, andmixtures thereof, interferons, GnRH agonists like buserelin, goserelin,leuprorelin (leuprolide), triptorelin, GnRH antagonists,bisphosphonates, glucagon-like peptides 1 and 2 and analogues such asGLP-1 receptor agonists and GLP-2 receptor agonists, GLP-1(7-37),GLP-1(7-36)amide, liraglutide, lixisenatide (AVE0010), and exenatide.

The pre-formulation is administrable by injection;

The pre-formulation is administrable by spraying, dipping, rinsing,application from a pad or ball roller, painting, dropping, aerosolspraying or pump spraying;

The pre-formulation has a turbidity factor of below 1, where theturbidity factor (TF) is defined as the absorbance (or turbidity) at 600nm of the aqueous phase resulting from placing a 200 mg aliquot ofpre-formulation in 5 ml of a 0.1 wt. % solution of sodium taurocholatein phosphate buffered saline (pH 7.4), at 37° C. for 6 hours under 150rpm rotation.

The pre-formulation is injectable and forms a depot providing continuousrelease of active agent for at least two weeks, preferably at least onemonth, wherein said active agent comprises at least one selected from:

-   -   a. leuprolide    -   b. octreotide;    -   c. GLP-1;    -   d. buprenorphine    -   e. fentanyl;    -   f. pasireotide;    -   g. goserelin.

In combination with the features and preferred features indicatedherein, the method(s) of delivery of the present invention may have oneor more of the following preferred features independently or incombination:

The method comprises the administration of at least one formulation withone or more preferred features as indicated above;

The method comprises the administration of at least one pre-formulationas described herein by subcutaneous injection, intramuscular injection,intra-cavity injection through tissue, intra-cavity injection into anopen cavity without tissue penetration, spraying, rolling, wiping,dabbing, painting, rinsing, or dropping;

The method comprises administration by means of a pre-filledadministration device as indicated herein;

The method comprises administration through a needle no larger than 20gauge, preferably smaller than 20 gauge, and most preferably 23 gauge orsmaller;

The method comprises a single administration every 7 to 360 days,preferably 7 to 120 days, for example 14 to 90 days;

The method comprises a single administration every 14 to 180 days,preferably around 90 days.

In combination with the features and preferred features indicatedherein, the use(s) of the pre-formulations indicated herein in themanufacture of medicaments may have one or more of the followingpreferred features independently or in combination:

The use comprises the use of at least one formulation with one or morepreferred features as indicated above;

The use comprises the manufacture of a medicament for administration ofat least one formulation as indicated herein;

The use comprises the manufacture of a medicament for administration bymeans of a pre-filled administration device as indicated herein;

The use comprises the manufacture of a medicament for administrationthrough a needle no larger than 20 gauge, preferably smaller than 20gauge, and most preferably 23 gauge or smaller;

The use comprises the manufacture of a medicament for administrationonce every 7 to 360 days, preferably 7 to 120 days, for example 14 to 90days.

In combination with the features and preferred features indicatedherein, the pre filled devices of the invention may have one or more ofthe following preferred features independently or in combination:

They contain a preferred formulation as indicated herein;

They comprise a needle smaller than 20 gauge, preferably no larger than23 gauge;

In combination with the features and preferred features indicatedherein, the method(s) of treatment of the present invention may have oneor more of the following preferred features independently or incombination:

The method comprises the administration of at least one formulation withone or more preferred features as indicated above;

The method is for the treatment of a condition selected from bacterialinfection; fungal infection; addiction to opioids, cocaine oramphetamine; cachexia; emetia; driving sickness; acromegaly; type I ortype II diabetes mellitus, and complications thereof, e.g. angiopathy,diabetic proliferative retinopathy, diabetic macular edema, nephropathy,neuropathy and dawn phenomenon, and other metabolic disorders related toinsulin or glucagon release, e.g. obesity, e.g. morbid obesity orhypothalamic or hyperinsulinemic obesity; enterocutaneous andpancreaticocutaneous fistula; irritable bowel syndrome; inflammatorydiseases, e.g. Grave's Disease, inflammatory bowel disease, psoriasis orrheumatoid arthritis; polycystic kidney disease; dumping syndrome;watery diarrhea syndrome; AIDS-related diarrhea; chemotherapy-induceddiarrhea; acute or chronic pancreatitis and gastrointestinal hormonesecreting tumors (e.g. GEP tumors, for example vipomas, glucagonomas,insulinomas, carcinoids and the like); lymphocyte malignancies, e.g.lymphomas or leukemias; prostate cancer; breast cancer; precociouspuberty; endometriosis; hepatocellular carcinoma; as well asgastrointestinal bleeding, e.g variceal oesophagial bleeding.

The method is for prophylaxis against at least one condition selectedfrom infection during surgery, infection during implantation, sunburn,infection at the site of burns, cuts or abrasions, oral infections,genital infections and infections resulting from activities resulting inexposure to infective agents.

The Invention will now be further illustrated by reference to thefollowing non-limiting Examples and the attached Figures.

FIGURES

FIG. 1: Apparent absorbance (turbidity) of the aqueous phase measured at600 nm for gels with the indicated lipid compositions (wt %) incubatedin 0.1 wt % sodium taurocholate (NaTC). The gels were incubated at 37°C. for 6 hours with moderate shaking (150 rpm). See also Table 1 forcomposition details.

FIG. 2: X-ray diffraction patterns of fully hydrated DOPE/GDO mixturesin saline at 25, 37 and 42° C. between DOPE/GDO weight ratios of 75/25and 35/65 as indicated in the figure. The relative diffraction peakpositions indicate the liquid crystalline structure change from reversedhexagonal to reversed micellar cubic (space group Fd3m) when the GDOcontent is increased.

FIG. 3: X-ray diffraction patterns of fully hydrated DOPE/GDO (60/40 byweight) and DOPE/TOC (60/40 by weight) mixtures in saline at 25, 37 and42° C. The relative diffraction peak positions indicate the samereversed micellar cubic (Fd3m) liquid crystalline structure within thetemperature range investigated.

FIG. 4: X-ray diffraction patterns of fully hydrated (in saline (0.9%NaCl w/v)) DOPE/GDO (50/50 by weight) mixtures including octreotide at25, 37 and 42° C. The octreotide concentration in the respective lipidformulation is indicated in the figure. The relative diffraction peakpositions indicate the same reversed micellar cubic (Fd3m) liquidcrystalline structure within the octreotide concentration andtemperature range investigated.

FIG. 5: In vivo pharmacokinetic profile of buprenorphine aftersubcutaneous administration of three formulations of the invention inrats. Error bars denote standard deviation (n=6). Formulationcompositions are provided in Example 12.

FIG. 6: In vivo pharmacokinetic profile of leuprolide (LEU) aftersubcutaneous administration in rats. Error bars denote standarddeviation (n=8). Formulation compositions are provided in Example 13.

FIG. 7: In vivo pharmacokinetic profile of octreotide (OCT) aftersubcutaneous administration in rats. Error bars denote standarddeviation (n=6). Formulation compositions are provided in Example 14.

FIG. 8: In vivo pharmacokinetic profile of octreotide (OCT) aftersubcutaneous administration in rats. Error bars denote standarddeviation (n=6). Formulation compositions are provided in Example 15.

FIG. 9: In vivo pharmacokinetic profile of octreotide (OCT) aftersubcutaneous administration in rats. Error bars denote standarddeviation (n=6). Formulation compositions are provided in Example 16.

FIG. 10: A comparison of the mechanical robustness of liquid crystallinegels formed by DOPE/GDO and SPC/GDO mixtures in aqueous solution (PBS,pH 7.4). The following phospholipid/GDO weight ratios were investigatedand compared: 70:30 (a), 65:35 (b), 60:40 (c), 55:45 (d) and 50:50 (e).

EXAMPLES Materials

Soy phosphatidylcholine (SPC)—Lipoid S100 from Lipoid, Germany

Dioleoylphosphatidylethanolamine (DOPE)—Lipoid PE 18:1/18:1 from Lipoid,Germany

Glycerol dioleate (GDO)—Rylo DG19 Pharma from Danisco, Denmark

α-Tocopherol (TOC)—from DSM, Switzerland

Ethanol (EtOH) 99.5% Ph. Eur.—from Solveco, Sweden

Sodium taurocholate (NaTC)—from Sigma-Aldrich, Sweden

Buprenorphine base (BUP)—from Jansen, Belgium

Leuprolide acetate (LEU)—from PolyPeptide Labs., USA

Octreotide hydrochloride (OCT)—from PolyPeptide Labs., USA

Pasireotide (SOM230) pamoate salt—from Novartis Pharma, Switzerland

Exenatide (EXT)—from Bachem, Switzerland

Goserelin acetate (GOS)—from PolyPeptide Labs., USA

Propylene Glycol (PG)—from Dow, Germany

Water for Injection (WFI)—from B. Braun, Germany

Example 1 Liquid Pre-Formulations Comprising Phospholipid andDiacylglycerol

Liquid pre-formulations (2 g) of phospholipid and diacylglycerol wereprepared by weighing in the respective lipid and solvent componentsaccording to Table 1 in 3 mL (2R) vials followed by roller mixing at 40°C. until homogenous liquid solutions were obtained (<20 h). Aftercooling to room temperature, all formulations were observed to behomogenous liquids of low viscosity.

TABLE 1 Composition of liquid pre-formulations comprising phospholipidand diacylglycerol (wt %) Formula- tion# SPC DOPE GDO EtOH Lipidcomposition (wt %) 1 45 — 45 10 SPC/GDO = 50/50 2 33.5 11.5 45 10SPC/DOPE/GDO = 37.5/12.5/50 3 22.5 22.5 45 10 SPC/DOPE/GDO = 25/25/50 411 34 45 10 SPC/DOPE/GDO = 12.5/37.5/50 5 — 45 45 10 DOPE/GDO = 50/50 652.8 — 35.2 12 SPC/GDO = 60/40 7 26.4 26.4 35.2 12 SPC/DOPE/GDO =30/30/40 8 — 52.8 35.2 12 DOPE/GDO = 60/40 9 — 36 54 10 DOPE/GDO = 40/6010 — 59.5 25.5 15 DOPE/GDO = 70/30

Example 2 Gelling of Pre-Formulations in Phosphate-Buffered Saline (PBS)

All liquid pre-formulations in Table 1 were subjected to a gelling testwhereby 0.20 g of the respective formulation was injected into 5 mL ofPBS (pH 7.4) in 6 mL (6R) injection glass vials using disposable 1 mLLuer-Lock syringes and 23 G needles. All formulations were easilyinjected using the 23 G needle size. The resulting gels were inspectedvisually after 1 h at room temperature and found to form coherent gelsthat could not be disrupted by mild shaking of the vials.

Example 3 Robustness of Lipid Gels in the Presence of Bile Salt

For long-term depot formulations and/or for per-oral formulations, acrucial property is related to the robustness of the gel towardserosion/fragmentation by endogenous surfactants and/or lipid-degradingenzymes. A way to study the robustness in vitro is to subject the lipidgels to a surfactant-rich aqueous environment and subsequently measurethe increased turbidity (or apparent absorbance) of the aqueous phaseresulting from surfactant-eroded lipid fragments. Such lipid fragmentsgive rise to substantial increase in solution turbidity due to lightscattering. Bile salts are often used as the surfactant of choice forstudying formulation dissolution given their biological relevance andendogenous nature. Accordingly, gels (0.20 g) formed in PBS by theformulations given in Table 1 were placed in 5 mL of a 0.1 wt % sodiumtaurocholate (NaTC) solution in PBS. The resulting samples werethereafter transferred to an incubator held at 37° C. with 150 rpmrotating speed. After 6 hours, the samples were taken out from theincubator, turned up-side down twice, and the respective aqueoussolution was transferred to a disposable semi-micro 1.5 mL cuvette forabsorbance measurement. The (apparent) absorbance or turbidity wasmeasured using a PerkinElmer Lambda 40 UV/Vis Spectrometer and air onlywas used for background correction. The results of the robustness studyare shown in FIG. 1.

As is evident from FIG. 1, the more of the PE-component (DOPE) that isincluded in the formulation, the more robust the gel is towardssurfactant-induced erosion. For example, by including 50% DOPE withrespect to SPC(SPC/DOPE=50/50 wt/wt) (Formulation#3 and 7 in Table 1), asignificant drop in turbidity is observed as a result of increasedrobustness towards surfactant-induced erosion. This effect is even morepronounced for formulations having an SPC/DOPE weight ratio of 25/75(Formulation#4) and most pronounced for formulations comprising only theDOPE component in combination with GDO (Formulation#5, 8, 9 and 10 inTable 1). In fact, the aqueous solutions of the gels comprising onlyDOPE/GDO (Formulation#5, 8, 9 and 10 in Table 1) were completelytransparent to the naked eye

Example 4 Liquid Pre-Formulations Comprising Phospholipid,Diacylglycerol and Buprenorphine

To 0.475 g of Formulation#1-10 in Table 1 (Example 1) was added 25 mgbuprenorphine base (BUP) to give 5 wt % BUP in total and the resultingsamples (in 2R injection glass vials) were placed on a roller mixer at40° C. for about 20 hours. All formulations were found to be homogenousand transparent low viscosity liquids after cooling to room temperature.

Example 5 Liquid Pre-Formulation Comprising Phospholipid, Diacylglyceroland Leuprolide Acetate

To 0.485 g of Formulation#5 in Table 1 (Example 1) was added 15 mgLeuprolide acetate (LEU) to give 3 wt % LEU in total and the resultingsample (in 2R injection glass vial) was placed on a roller mixer at roomtemperature for about 48 hours.

Example 6 Liquid Preformulations Comprising Phospholipid,Diacylglycerol, Low Viscosity Organic Solvent and Polar Solvent

Liquid pre-formulations (1 g) of phospholipid and diacylglycerol wereprepared as described in Example 1. After mixing, all formulations wereobserved to be homogenous liquids of low viscosity at room temperature.The compositions of the formulations are given in Table 2.

TABLE 2 Composition of liquid pre-formulations comprising phospholipid,diacylglycerol, low viscosity organic solvent and polar solvent (wt %)Formulation# DOPE GDO EtOH PG WFI 11 35.3 53.0 9.8 1.9 — 12 34.6 51.99.6 3.9 — 13 34.1 51.1 9.5 5.3 — 14 32.6 49.0 9.2 9.2 — 15 51.8 34.511.8 — 1.9 16 50.9 33.9 11.6 — 3.6 17 49.9 33.3 11.4 — 5.4 18 48.2 32.211.0 — 8.6

Example 7 Liquid Pre-Formulations Comprising Phospholipid andα-Tocopherol

Liquid pre-formulations (2 g) of phospholipid and α-tocopherol (TOC) areprepared by weighing in the respective lipid and solvent componentsaccording to Table 3 in 3 mL (2R) vials followed by roller mixing at 40°C. until homogenous liquid solutions are obtained (<20 h). After coolingto room temperature, all formulations are observed to be homogenousliquids of low viscosity.

TABLE 3 Composition of liquid pre-formulations comprising phospholipidand a-tocopherol (TOC) (wt %) Form- ulation# SPC DOPE TOC EtOH Lipidcomposition (wt %) 19 33.5 11.5 45 10 SPC/DOPE/TOC = 37.5/12.5/50 2022.5 22.5 45 10 SPC/DOPE/TOC = 25/25/50 21 11   34 45 10 SPC/DOPE/TOC =12.5/37.5/50 22 — 45 45 10 DOPE/TOC = 50/50 23 26.4 26.4 35.2 12SPC/DOPE/TOC = 30/30/40 24 — 52.8 35.2 12 DOPE/TOC = 60/40 25 — 36 54 10DOPE/TOC = 40/60

Example 8 Liquid Crystalline Phase Structures from DOPE/GDO Mixtures inthe Presence of Aqueous Phase

Liquid pre-formulations (2 g) of DOPE and GDO were prepared by weighingthe required amount of the respective lipid components in 3 mL (2R)vials followed by addition of EtOH at a total concentration of 10-15 wt%. The weight ratio of the lipids in the different samples was in therange DOPE:GDO=75:25-35:65. The samples were roller mixed at 40° C.until homogenous liquid solutions were obtained (<20 h). After coolingto room temperature, all formulations were observed to be homogenousliquids of low viscosity. The respective formulation (0.5 g) wasthereafter injected into 5 mL of saline (0.9% w/v NaCl) in 6 mL (6R)injection glass vials using disposable 1 mL Luer-Lock syringes and 23 Gneedles. All formulations were easily injected using the 23 G needlesize. The resulting gels were allowed to equilibrate on a roller mixerat ambient room temperature for 10 days before small angle X-rayscattering (SAXS) measurements.

Synchrotron SAXS measurements were performed at the 1911 beamline atMAX-lab (Lund University, Sweden), using a Marresearch 165 mm CCDdetector mounted on a Marresearch Desktop Beamline baseplate. TheDOPE/GDO/saline liquid crystalline samples were mounted between kaptonwindows in a steel sample holder at the sample-to-detector distance of1916.8 mm. Diffractograms were recorded at the indicated temperatures(FIG. 2) under high vacuum with a wavelength of 0.91 Å and the beam sizeof 0.25×0.25 mm (full width at the half maximum) at the sample. Theexposure time for each sample was 3 min. The resulting CCD images wereintegrated and analysed using calibrated wavelengths and detectorpositions. The relative diffraction peak positions shown in FIG. 2indicate that the liquid crystalline structure changes from reversedhexagonal (H2) at high DOPE content to reversed micellar cubic (12,space group Fd3m) when the GDO content is increased.

Example 9 Liquid Crystalline Phase Structures from DOPE/TOC and DOPE/GDOMixtures in the Presence of Aqueous Phase

Liquid pre-formulations (2 g) of DOPE/GDO and DOPE/TOC were prepared byweighing the required amount of the respective lipid components in 3 mL(2R) vials followed by addition of EtOH at a total concentration of 10wt %. The weight ratio of the lipids in the different samples wasDOPE:GDO and DOPE:TOC=60:40. The samples were roller mixed at 40° C.until homogenous liquid solutions were obtained (<20 h). After coolingto room temperature, the formulations were observed to be homogenousliquids of low viscosity. The respective formulation (0.5 g) wasthereafter injected into 5 mL of saline (0.9% w/v NaCl) in 6 mL (6R)injection glass vials using disposable 1 mL Luer-Lock syringes and 23 Gneedles. The formulations were easily injected using the 23 G needlesize. The resulting gels were allowed to equilibrate on a roller mixerat ambient room temperature for 10 days before small angle X-rayscattering (SAXS) measurements.

Synchrotron SAXS measurements were performed as described in Example 8and the results are shown in FIG. 3. The relative diffraction peakpositions (FIG. 3) indicate the same reversed micellar cubic (Fd3m)liquid crystalline structure for both DOPE/GDO and DOPE/TOC (60/40wt/wt) mixtures within the temperature range investigated.

Example 10 Liquid Crystalline Phase Structures from DOPE/GDOPreformulations Comprising Octreotide in the Presence of Aqueous Phase

Liquid pre-formulations (5 g) comprising DOPE and GDO were prepared byweighing the required amount of the respective lipid component in 10 mL(10R) vials followed by addition of EtOH. The samples were roller mixedat 40° C. until homogenous liquid solutions were obtained (<20 h). Aftercooling to room temperature, octreotide hydrochloride (OCT) was added tothe formulations at concentrations of 30 and 45 mg OCT free base/mL,respectively, followed by magnetic stirring until the formulations wereobserved to be homogenous liquids of low viscosity. The respectiveformulation (0.5 g) was thereafter injected into 5 mL of saline (0.9%w/v NaCl) in 6 mL (6R) injection glass vials using disposable 1 mLLuer-Lock syringes and 23 G needles. The formulations were easilyinjected using the 23 G needle size. The resulting gels were allowed toequilibrate on a roller mixer at ambient room temperature for 10 daysbefore small angle X-ray scattering (SAXS) measurements. The finalcompositions of the preformulations comprising OCT are provided in Table4.

TABLE 4 Composition of liquid pre-formulations comprising DOPE, GDO,EtOH and OCT (wt %) Form- ulation# OCT DOPE GDO EtOH Comment 26 3.6243.19 43.19 10.00 Corresponding to 30 mg octreotide free base per mLwhen corrected for peptide purity and content and formulation density.27 5.43 42.29 42.29 10.00 Corresponding to 45 mg octreotide free baseper mL when corrected for peptide purity and content and formulationdensity.

Synchrotron SAXS measurements were performed as described in Example 8and the results are shown in FIG. 4 where also the diffractogram for theDOPE/GDO mixture without octreotide is included. The relativediffraction peak positions indicate that the reversed micellar cubic(Fd3m) liquid crystalline structure observed for the DOPE/GDO mixturewithout the octreotide active agent is retained within the octreotideconcentration and temperature range investigated.

Example 11 Formulation Comprising DOPE, GDO, EtOH, PG and Pasireotide(Pamoate Salt)

A liquid pre-formulation (2 g) comprising DOPE and GDO was prepared byweighing the required amount of the respective lipid component in 2 mL(2R) vials followed by addition of the required amount of EtOH and PG.The sample was roller mixed at 40° C. until a homogenous liquid solutionwas obtained (<20 h). After cooling to room temperature, pasireotidepamoate (or SOM230) was added to the formulation to give a finalconcentration of ca 30 mg/mL pasireotide (calculated as free base). Thefinal sample composition is given in Table 5.

TABLE 5 Composition of liquid pre-formulation comprising DOPE, GDO,EtOH, PG and Pasireotide (wt %). The pasireotide concentrationcorresponds to approximately 30 mg pasireotide free base/mL. PasireotideFormulation# pamoate DOPE GDO EtOH PG 28 4.77 38.50 38.76 8.58 9.39

Example 12 In Vivo Pharmacokinetic Study of Formulations ComprisingBuprenorphine

Liquid pre-formulations (6 g) comprising BUP, DOPE and GDO were preparedby weighing the required amount of the respective component in 10 mL(10R) vials followed by addition of EtOH. The samples were roller mixedat 40° C. until homogenous liquid solutions were obtained (ca 6 h). Therespective formulation was thereafter sterile filtered under 2.5 barnitrogen pressure using a sterile 0.2 micron PVDF membrane filter fromMillipore. The formulation compositions are provided in Table 6.

TABLE 6 Composition of liquid pre-formulations comprising DOPE, GDO,EtOH and BUP (wt %). The BUP concentration corresponds to 50 mg BUPbase/mL. Formulation# BUP DOPE GDO EtOH BUP-1 5.29 50.83 33.88 10.00BUP-2 5.29 42.36 42.36 10.00 BUP-3 5.29 33.88 50.83 10.00

The formulations in Table 6 were injected subcutaneously to maleSprague-Dawley rats at a dose volume of 0.2 mL/kg (10 mg BUP/kg). Bloodfor pharmacokinetics were collected pre-dose, and 1 hour, 6 hours, 1day, 2 days, 5 days, 8 days, 14 days, 21 days and 28 days after dosing.Blood samples of 0.2 mL were collected by sub-lingual bleeding intoEDTA-treated test tubes (Capiject 3T-MQK, Terumo Medical Corporation).The blood was placed on ice immediately after collection and centrifuged(approximately 1500×g, at 5° C. for 10 min) within 30 to 60 minutes. Theplasma was transferred into properly labelled translucent 1.5-mLpropylene test tubes (Microcentrifuge tubes, Plastibrand, Buch & Holm)and stored below −70° C. until transportation on dry ice for analysis.The buprenorphine concentration in the rat plasma samples were analysedusing an ELISA assay for determination of BUP in EDTA rat plasmasamples.

The obtained PK profiles are shown in FIG. 5 demonstrating sustainedrelease of BUP over at least 28 days.

Example 13 In Vivo Pharmacokinetic Study of Formulations ComprisingLeuprolide Acetate

Liquid pre-formulations comprising phospholipid and GDO were prepared byweighing the required amount of the respective lipid component in 15 mL(15R) vials followed by addition of EtOH. The samples were roller mixedat 40° C. until homogenous liquid solutions were obtained. The requiredamount of LEU was dissolved in the required amount of WFI containing 0.1mg EDTA/mL. The respective lipid/EtOH solution was thereafter added tothe LEU/WFI solution. The resulting formulations were finally rollermixed at ambient RT and subjected to sterile filtration under 2.5 barnitrogen pressure using a sterile 0.2 micron PVDF membrane filter fromMillipore. The total batch size was 7 g and the final formulationcompositions are provided in Table 7.

TABLE 7 Composition of liquid pre-formulations comprising phospholipid,GDO, co-solvent and LEU (wt %). The LEU concentration corresponds to 25mg leuprolide acetate/mL. Formula- tion# LEU DOPE SPC GDO EtOH WFI*LEU-1 2.70 — 37.60 37.69 11.99 10.02 LEU-2 2.70 19.32 19.31 38.62 10.0210.04 *Containing 0.1 mg EDTA (disodium)/mL

The formulations in Table 7 were injected subcutaneously to maleSprague-Dawley rats at a dose volume of 0.2 mL/kg (5 mg LEU acetate/kg).Blood for pharmacokinetics was collected at pre-dose, and 1 hour, 6hours, 1 day, 2 days, 5 days, 8 days, 14 days, 21 days and 28 days afterdosing. Blood samples of 0.25 mL were collected by sub-lingual bleedinginto EDTA-treated test tubes (Capiject 3T-MQK, Terumo MedicalCorporation). The blood was placed on ice immediately after collectionand centrifuged (approximately 1500×g, at 5° C. for 10 min) within 30 to60 minutes. The plasma was transferred into properly labelled green1.5-mL propylene test tubes (Microcentrifuge tubes, Plastibrand, Buch &Holm) and stored below −70° C. until transportation on dry ice foranalysis. Analysis of Leuprolide was performed using the (Des-Gly10,D-LEU6, Pro-NHEt9)-LHRH (Leuprolide) high sensitivity EIA kit (S-1174,Bachem/Peninsula Laboratories) adapted for analysis of LEU in rat EDTAplasma.

The obtained PK profiles are shown in FIG. 6 demonstrating sustainedrelease of LEU over at least 28 days for both formulations. Notably, theLEU-2 formulation comprising DOPE showed more stable plasma levels overtime and in particular higher plasma levels from day 14 through day 28.

Example 14 In Vivo Pharmacokinetic Study 1 of Formulations ComprisingOctreotide

Liquid pre-formulations comprising DOPE/GDO and SPC/GDO were prepared byweighing the required amount of the respective lipid component in 15 mL(15R) vials followed by addition of EtOH. The samples were roller mixedat 40° C. until homogenous liquid solutions were obtained. The requiredamount of octreotide hydrochloride was weighed into a 10 mL (10R) glassvial followed by addition of the respective lipid/EtOH solution. Theresulting formulations were roller mixed at ambient RT until homogenousliquid solutions were obtained. The respective formulation wasthereafter sterile filtered under 2.5 bar nitrogen pressure using asterile 0.2 micron PVDF membrane filter from Millipore. The batch sizewas 7 g and the final formulation compositions are provided in Table 8.

TABLE 8 Composition of liquid pre-formulations comprising phospholipid,GDO, co-solvent and OCT (wt %). The OCT concentration corresponds to 45mg octreotide free base/mL. Formula- tion# OCT DOPE SPC GDO EtOH OCT-15.43 42.29 — 42.29 10.00 OCT-2 5.43 — 42.29 42.29 10.00

The formulations in Table 8 were injected subcutaneously to maleSprague-Dawley rats at a dose volume of 0.6 mL/kg (27 mg octreotide freebase/kg). Blood for pharmacokinetics were collected pre-dose, and 1hour, 6 hours, 1 day, 2 days, 5 days, 8 days, 14 days, 21 days, 28 daysand 35 days after dosing. Blood samples of 0.25 mL were collected bysub-lingual bleeding into EDTA-treated test tubes (Capiject 3T-MQK,Terumo Medical Corporation). The blood was placed on ice immediatelyafter collection and centrifuged (approximately 1500×g, at 5° C. for 10min) within 30 to 60 minutes. The plasma was transferred into properlylabelled blue 1.5-mL propylene test tubes (Microcentrifuge tubes,Plastibrand, Buch & Holm) and stored below −70° C. until transportationon dry ice for analysis. The plasma samples were analysed with the ELISAkit S-1275 (Bachem/Peninsula Laboratories) “Octreotide—EIA Kit, Host:Rabbit, High Sensitivity”, adapted for analysis of OCT in rat EDTAplasma.

The obtained PK profiles are shown in FIG. 7 demonstrating sustainedrelease of OCT over at least 35 days for both formulations. Notably, theOCT-1 formulation comprising DOPE showed more stable plasma levels overtime and in particular higher plasma levels from day 14 through day 35.

Example 15 In Vivo Pharmacokinetic Study 2 of Formulations ComprisingOctreotide

Liquid pre-formulations (5 g) comprising phospholipid, GDO, co-solventsand octreotide were prepared as described in Example 14. The finalformulation compositions are provided in Table 9.

TABLE 9 Composition of liquid pre-formulations comprising phospholipid,GDO, co-solvent and OCT (wt %) Formula- tion# OCT DOPE SPC GDO EtOH PGComment OCT-1 5.43 42.29 — 42.29 10.00 — 45 mg OCT free base/mL OCT-25.43 — 42.29 42.29 10.00 — 45 mg OCT free base/mL OCT-3 2.40 43.80 —43.80 10.00 — 20 mg OCT free base/mL OCT-4 2.39 — 42.31 42.31  6.50 6.5020 mg OCT free base/mL

The formulations in Table 9 were injected subcutaneously to maleSprague-Dawley rats at a dose volume of 0.2 mL/kg (9 mg OCT free base/kgfor OCT-1 and OCT-2 and 4 mg OCT free base/kg for OCT-3 and OCT-4).Blood for pharmacokinetics were collected pre-dose, and 1 hour, 6 hours,1 day, 4 days, 6 days, 8 days, 11 days, 14 days, 18 days, 21 days, 25days and 28 days after dosing. The sampling procedure and bioassay wereas described in Example 14.

The obtained PK profiles are shown in FIG. 8 demonstrating sustainedrelease of OCT over at least 28 days for all formulations. Notably, theOCT-1 and OCT-3 formulations comprising DOPE showed more stable plasmalevels over time and in particular higher plasma levels from day 14through day 28. The variability in measured plasma concentrations atlonger times post injection (≧21 days) were also lower for the DOPEbased formulations, especially pronounced for the OCT-3 formulation with20 mg OCT free base/mL.

An interesting and noticeable finding in the study was that depots ofthe DOPE-based formulations were present at the injection site in allanimals at termination whereas half or more of the animals receiving theSPC-based formulations showed complete clearance of the depot matrix.This indicates differences in lipid matrix in vivo degradation kineticsand supports the PK data at longer times post injection where theDOPE-based formulations showed higher and less variable plasma levels.

Example 16 In Vivo Pharmacokinetic Study 3 of Formulations ComprisingOctreotide

Liquid pre-formulations (5 g) comprising phospholipid, GDO, co-solventsand octreotide were prepared as described in Example 14. The finalformulation compositions are provided in Table 10.

TABLE 10 Composition of liquid pre-formulations comprising phospholipid,GDO, co-solvent and OCT (wt %). The OCT concentration corresponds to 20mg OCT free base/mL. Formulation# OCT DOPE SPC GDO EtOH OCT-3 2.40 43.80— 43.80 10.00 OCT-5 2.39 35.04 — 52.57 10.00 OCT-6 2.39 52.57 — 35.0410.00 OCT-7 2.39 39.43 4.37 43.81 10.00 OCT-8 2.39 35.05 8.75 43.8110.00

The formulations in Table 10 were injected subcutaneously to maleSprague-Dawley rats at a dose volume of 0.2 mL/kg (4 mg OCT freebase/kg). Blood for pharmacokinetics were collected pre-dose, and 1hour, 6 hours, 1 day, 4 days, 6 days, 8 days, 12 days, 14 days, 19 days,21 days and 28 days after dosing. The sampling procedure and bioassaywere as described in Example 14.

The obtained PK profiles are shown in FIG. 9 demonstrating sustainedrelease of OCT over at least 28 days for all formulations. A higherinitial release and lower plasma levels of OCT were observed for theOCT-5 formulation whereas the plasma profiles were similar for the otherformulations.

Example 17 Formulations Comprising DOPE, GDO, EtOH, PG and GLP-1Receptor Agonists

Liquid pre-formulations (2 g) comprising DOPE and GDO are prepared byweighing the required amount of the respective lipid component in 2 mL(2R) vials followed by addition of the required amount of EtOH and PG.The samples are roller mixed at 40° C. until homogenous liquid solutionsare obtained. After cooling to room temperature, exenatide (EXT) andliraglutide (LIR), respectively, are added to the formulations to give afinal concentration of approximately 10 mg GLP-1 receptor agonist/mL.The final sample compositions are given in Table 11.

TABLE 11 Composition of liquid pre-formulations comprising DOPE, GDO,EtOH, PG and EXT or LIR (wt %). The EXT/LIR concentration corresponds toapproximately 10 mg peptide/mL. EXT LIR DOPE GDO EtOH PG 1.25 — 39.9240.19 8.90 9.73 — 1.25 39.40 39.32 10.02 10.01

Example 18 Mechanical Robustness of Liquid Crystals Formed by DOPE/GDOand SPC/GDO Mixtures in Aqueous Solution

Liquid pre-formulations (1 g) of DOPE/GDO and SPC/GDO mixtures wereprepared by weighing the required amount of the respective lipidcomponents in 3 mL (2R) vials followed by addition of EtOH at a totalconcentration of 10 wt %. The weight ratio of the lipids in thedifferent samples was in the range DOPE:GDO=70:30-50:50 andSPC:GDO=70:30-50:50. The samples were roller mixed at 40° C. untilhomogenous liquid solutions were obtained (<20 h). After cooling to roomtemperature, the formulations were observed to be homogenous liquids oflow viscosity. The respective formulation (0.5 g) was thereafterinjected into 5 mL of phosphate buffered saline (pH 7.4) in 10 mL (10R)injection glass vials using disposable 1 mL Luer-Lock syringes and 23 Gneedles. The formulations were easily injected using the 23 G needlesize. The resulting gels were allowed to equilibrate on a mechanicalmixing table at 37° C. and 150 rpm for 20 days before robustnessmeasurements.

The liquid crystalline robustness measurements were performed by usingTA.XT plus Texture Analyzer (Stable Micro Systems Ltd., UK) equippedwith a 2 mm thick stainless needle. Force vs. distance dependencies wereregistered by penetrating the needle about 4 mm into the liquidcrystalline gels at a speed of 0.5 mm/s. The higher the force requiredto penetrate the needle, the higher the mechanical resistance of thegel.

The results are shown in FIG. 10 showing in all cases that theDOPE-based liquid crystalline (LC) gels are significantly moremechanically robust compared to the SPC-based LC gels. This result is inline with the higher resistance towards surfactant-induced erosion asexemplified in Example 1. The higher mechanical robustness of theDOPE-based formulations in comparison to SPC-based formulations may alsobe one reason for the difference in in vivo performance between theformulation types as described in Examples 13-15.

Example 19 Liquid Pre-Formulations Comprising Phospholipid,Diacylglycerol and Goserelin Acetate

Liquid pre-formulations (2 g) comprising DOPE, SPC and GDO are preparedby weighing the required amount of the respective lipid component in 2mL (2R) vials followed by addition of the required amount of co-solvent.The samples are roller mixed at 40° C. until homogenous liquid solutionsare obtained. After cooling to room temperature, goserelin acetate (GOS)is added to the formulations to the final concentration indicated inTable 12.

TABLE 12 Composition of liquid pre-formulations comprising DOPE, SPC,GDO, co-solvent and GOS (wt %). GOS DOPE SPC GDO EtOH PG WFI* 1.50 44.25— 44.25 10.00 — — 2.70 43.65 — 43.65 10.00 — — 1.50 48.68 — 39.82 10.00— — 2.70 48.02 — 39.28 10.00 — — 1.50 39.82 4.43 44.25 10.00 — — 1.5035.40 8.85 44.25 10.00 — — 1.50 41.75 — 41.75 7.50 7.50 — 2.70 41.15 —41.15 7.50 7.50 — 1.50 39.25 — 39.25 10.00 10.00 — 2.70 38.65 — 38.6510.00 10.00 — 1.50 40.25 — 40.25 12.00 — 6.00 2.70 39.65 — 39.65 12.00 —6.00

The invention claimed is:
 1. A pre-formulation comprising a lowviscosity, non-liquid crystalline, mixture of: a) at least one diacylglycerol and/or at least one tocopherol; b) at least one phospholipidcomponent having i. polar head groups comprising more than 50%phosphatidyl ethanolamine, and ii. two acyl chains each independentlyhaving 16 to 20 carbons wherein at least one acyl chain has at least oneunsaturation in the carbon chain, and there are no more than fourunsaturations over two carbon chains; c) at least one biocompatible,oxygen containing, low viscosity organic solvent; wherein optionally atleast one bioactive agent is dissolved or dispersed in the low viscositymixture; wherein the pre-formulation has a viscosity of 0.1-5000 mPas;and wherein the pre-formulation forms, or is capable of forming, atleast one non-lamellar liquid crystalline phase structure upon contactwith an aqueous fluid.
 2. A pre-formulation as claimed in claim 1wherein said liquid crystalline phase structure is a reversed hexagonalphase structure or a reversed cubic phase structure or mixtures thereof.3. A pre-formulation as claimed in claim 2 where said liquid crystallinephase structure is selected from H₂, I₂, or mixtures thereof.
 4. Apre-formulation as claimed in claim 1 wherein the non-polar tail groupsof component a) each independently consist essentially of unsaturatedC18 groups.
 5. A pre-formulation as claimed in claim 1 wherein componenta) consists essentially of at least one tocopherol.
 6. A pre-formulationas claimed in claim 1 wherein component a) consists essentially of amixture of GDO and tocopherol.
 7. A pre-formulation as claimed in claim1 wherein component b) is selected from phosphatidyl ethanolamines, ormixtures of phosphatidyl ethanolamines with at least one selected fromphosphatidyl cholines, phosphatidyl inositols, and sphingomyelins.
 8. Apre-formulation as claimed in claim 1 wherein said phospholipidcomponent b) comprises at least 75% PE.
 9. A pre-formulation as claimedin claim 1 wherein the phospholipid component b) further comprises atleast one phospholipid having i. polar head groups comprising at least90% phosphatidyl choline, and ii. two acyl chains each independentlyhaving 16 to 20 carbons wherein at least one acyl chain has at least oneunsaturation in the carbon chain, and there are no more than fourunsaturations over two carbon chains.
 10. A pre-formulation as claimedin claim 1 wherein the phospholipid component b) comprises at least 10%PC SPC, DOPC or mixtures thereof.
 11. A pre-formulation as claimed inclaim 1 wherein component b) comprises more than 70% PE and less than30% PC.
 12. A pre-formulation as claimed in claim 1 having a molecularsolution, L₂ and/or L₃ phase structure.
 13. A pre-formulation as claimedin claim 1 having a ratio of a) to b) of between 80:20 and 5:95 byweight.
 14. A pre-formulation as claimed in claim 1 having at least 15%of component a) and/or at least 15% of component b) by weight ofcomponents a)+b)+c).
 15. A pre-formulation as claimed in claim 1 having2 to 40% component c) by weight of components a)+b)+c).
 16. Apre-formulation as claimed in claim 1 wherein component c) is selectedfrom an alcohol, a ketone, an ester, an ether, an amide amides, asulphoxide and mixtures thereof.
 17. A pre-formulation as claimed inclaim 1 wherein component c) comprises ethanol, NMP, DMSO or mixturesthereof.
 18. A pre-formulation as claimed in claim 1 additionallycomprising up to 10% by weight of a)+b) of a charged amphiphile.
 19. Apre-formulation as claimed in claim 1 having 0.1-10 wt. % of said activeagent by weight of components a)+b)+c).
 20. A pre-formulation as claimedin claim 1 further comprising: d) up to 20 wt. % of at least one polarsolvent by weight of components a)+b)+c)+d).
 21. A pre-formulation asclaimed in claim 20 wherein component d) comprises or consists of wateror propylene glycol or mixtures thereof.
 22. A pre-formulation asclaimed in claim 20 where component d) comprises at least 2% water. 23.A pre-formulation as claimed in claim 20 wherein component d) is presentat a level of 1.2 to 20% by weight.
 24. A pre-formulation as claimed inclaim 20 wherein component c) comprises solvent selected from the groupconsisting of ethanol, propanol, isopropanol or mixtures thereof, DMSO,NMP or mixtures of NMP and ethanol.
 25. A pre-formulation as claimed inclaim 20 wherein components c) and d) combined are present at a totallevel less than or equal to 40% by weight.
 26. A pre-formulation asclaimed in claim 1 wherein said active agent is selected from a drug, anantigen, a nutrient, a cosmetic, a fragrance, a flavoring, a diagnosticagent, a vitamin, a dietary supplement and mixtures thereof; wherein, ifpresent, said drug is selected from a hydrophilic small molecule drug, alipophilic small molecule drug, an amphiphilic small molecule drug, apeptide, a protein, an oligonucleotide and mixtures thereof.
 27. Apre-formulation as claimed in claim 26 wherein said drug is selectedfrom an opioid agonist, a GnRH agonist, a GnRH antagonists, asomatostatin, a somatostatin receptor (SSTR) agonist, a glucagon-likepeptide 1 (GLP-1) receptor agonist, liraglutide, exenatide,lixisenatide, a glucagon-like peptide 2 agonist, and mixtures thereof.28. A pre-formulation as claimed in claim 1 which is administrable byinjection, spraying, dipping, rinsing, application from a pad or ballroller, painting, dropping, aerosol spraying or pump spraying.
 29. Aninjectable form of the pre-formulation as claimed in claim 1, whereinthe pre-formulation comprises at least one bioactive agent that isdissolved or dispersed in the low viscosity mixture, and that isselected from: leuprolide; octreotide; GLP-1; buprenorphine; fentanyl;pasireotide; and goserelin.
 30. A pre-formulation as claimed in claim 1excluding the pre-formulations listed below: Pre- Level form- of ulationActive # Active agent Agent DOPE SPC GDO EtOH PG 26 Octreatide 3.6243.19 — 43.19 10.00 — Hydrochloride 27 Octreotide 5.43 42.29 — 42.2910.00 — Hydrochloride 28 Pasireotide 4.77 38.50 — 38.76 8.58 9.39Pamoate OCT-1 Octreotide 5.43 42.29 — 42.29 10.00 — Hydrochloride OCT-3Octreotide 2.40 43.80 — 43.80 10.00 — Hydrochloride OCT-5 Octreotide2.39 35.04 — 52.57 10.00 — Hydrochloride OCT-6 Octreotide 2.39 52.27 —35.04 10.00 — Hydrochloride OCT-7 Octreotide 2.39 39.43 4.37 43.81 10.00— Hydrochloride OCT-8 Octreotide 2.39 35.05 8.75 43.81 10.00 —Hydrochloride

wherein DOPE is dioleoylphosphatidyl ethanolamine, GDO is glyceroldioleate, SPC is Soy phosphatidyl choline, EtOH is ethanol, PG ispropylene glycol, and all figures are % by weight of the totalcomposition.
 31. A method of delivery of a bioactive agent to a human ornon-human animal body, this method comprising administering apre-formulation as claimed in claim 1 comprising a non-liquidcrystalline, low viscosity mixture of: a) at least one diacyl glyceroland/or at least one tocopherol; b) at least one phospholipid componenthaving i. polar head groups comprising more than 50% phosphatidylethanolamine, and ii. two acyl chains each independently having 16 to 20carbons wherein at least one acyl chain has at least one unsaturation inthe carbon chain, and there are no more than four unsaturations over twocarbon chains; c) at least one biocompatible, oxygen containing, lowviscosity organic solvent; and at least one bioactive agent is dissolvedor dispersed in the low viscosity mixture, whereby to form at least onenon-lamellar liquid crystalline phase structure upon contact with anaqueous fluid in vivo following administration; wherein saidpre-formulation is administered by a method selected from subcutaneousinjection, intramuscular injection, intra-cavity injection throughtissue, intra-cavity injection into an open cavity without tissuepenetration, spraying, rolling, wiping, dabbing, painting, rinsing, ordropping.
 32. A method for the preparation of a liquid crystallinecomposition comprising exposing a pre-formulation as claimed in claim 1to an aqueous fluid in vivo.
 33. A process for the formation of apre-formulation according to claim 1 suitable for the administration ofa bioactive agent to a subject, said process comprising forming anon-liquid crystalline, low viscosity mixture of a) at least one diacylglycerol and/or at least one tocopherol; b) at least one phospholipidcomponent having i. polar head groups comprising more than 50%phosphatidyl ethanolamine, and ii. two acyl chains each independentlyhaving 16 to 20 carbons wherein at least one acyl chain has at least oneunsaturation in the carbon chain, and there are no more than fourunsaturations over two carbon chains; c) at least one biocompatible,oxygen containing, low viscosity organic solvent; having a ratio of a)to b) of between 80:20 and 20:80 by weight; and dissolving or dispersingat least one bioactive agent in the low viscosity mixture, or in atleast one of components a, b or c prior to forming the low viscositymixture.
 34. A method of treatment or prophylaxis of a human ornon-human animal subject comprising administration of a pre-formulationas claimed in claim 1, wherein the condition to be treated is selectedfrom bacterial infection, fungal infection, skin soreness, eyeconditions, genital soreness, infections and conditions for the fingerand/or toe nails, travel sickness, addiction including nicotineaddiction, periodontal infection, conjunctivitis, glaucoma and hormonedeficiency or imbalance; or wherein the condition is for prophylaxisagainst at least one condition selected from infection during surgery,infection during implantation, sunburn, infection at the site of burns,cuts or abrasions, oral infections, genital infections and infectionsresulting from activities resulting in exposure to infective agents.